Rna ribozyme polymerases, dephosphorylases, restriction endoribo-nucleases and methods

ABSTRACT

RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribonsomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.

This is a divisional of Ser. No. 06/937,327, filed on Dec. 3, 1986,which is now U.S. Pat. No. 4,987,071.

This invention concerns compositions of RNA functioning as an RNAenzyme, i.e. a ribozyme in several capacities: dephosphorylase (acidphosphatase and transphosphorylase), ribonucleotidyl transferase(polymerase activity) and sequence-specific endoribonuclease activities.

SUMMARY

It is found that purified ribonucleic acid (RNA) can serve as an enzymeacting on other RNA molecules in vitro (ribozyme) as a: 1)dephosphorylating enzyme catalyzing the removal of 3' terminal phosphateof RNA in a sequence-specific manner, 2) RNA polymerase (nucleotidyltransferase) catalyzing the conversion of oligoribonucleotides topolyribonucleotides, 3) sequence specific endoribonuclease. (This latteractivity is also referred to as RNA restriction endonuclease orendoribonuclease activity.)

DESCRIPTION OF THE DRAWINGS

FIG. 1 compares RNA self-splicing (A) to RNA endoribonuclease activity(B).

FIG. 2 shows products of cleavage of a variety of RNA substrates by theRNA endoribonuclease.

FIG. 3 compares different substrate activity of three variant forms ofL-19 IVS ribozyme in 2.5M urea.

FIG. 4 shows the time course of oligonucleotide cleavage.

FIG. 5 shows the cleavage and reforming of oligoribonucleotidesubstrates by L-19 IVS RNA.

FIG. 6 shows the kinetics of conversion of pC₅ to larger and smalleroligonucleotides with L-19 IVS RNA.

FIG. 7 shows the enzyme-substrate intermediate of L-19 IVS RNA.

FIG. 8 is a model for enzymatic mechanism of L-19 IVS RNA acting as aribonucleotidyl transferase.

FIG. 9 shows the competitive inhibition of pC₅ reaction by dC₅.

FIG. 10 shows the relation of reactions catalyzed by the L-19 IVS RNA toself-splicing and related IVS RNA-mediated reactions.

FIG. 11 shows the dephosphorylation of oligo (cytidylic acid)3-phosphate.

FIG. 12 shows the effect of pH on phosphotransfer and nucleotidyltransfer reactions.

FIG. 13 shows that the phosphorylation of L-19 IVS RNA ribozyme isreversible.

FIG. 14 shows the L-19 IVS RNA acts catalytically as aphosphotransferase.

FIG. 15 shows the single active site model for the activity of L-19 IVSRNA on phosphate diester and phosphate monoester substrates

FIG. 16 shows the plasmid construction which produces the L-21 IVS RNA.

DETAILED DESCRIPTION OF THE DRAWINGS Figure Legends

FIG. 1 A model for the L-19 IVS RNA acting like an RNA restrictionendonuclease by a mechanism that is an intermolecular version of thefirst step of pre-rRNA self-splicing. Thin letters and lines representIVS sequences, boldface letters and thick lines represent exon sequences(above) or substrate RNA sequences (below), and the G in italics is afree guanosine nucleotide or nucleoside.

FIG. 2 The L-19 IVS-beta RNA cleaves large RNA substrates with transferof guanosine. a, Uniformly labeled 0.6 uM (u=micro) pAK105 RNA (508 nt)incubated with 0.2 uM L-19 IVS-beta RNA and 0, 0.1 or 0.5 mM GTP(unlabeled) for 1 h under conditions described below. (M) Mixture of 4substrate RNAs as molecular weight markers. b, Various tritiated RNAsubstrates (1 ug each) incubated with 0.2 uM L-19 IVS-beta RNA and 120uM [alpha-³² P]GTP for 1 h under the same reaction conditions as a.Autoradiogram reveals [³² p]GTP-labeled products only. The L-19 IVS-betaRNA also becomes GTP-labeled during the incubation. c, Nucleotidesequence of the cleavage product pAK105(1) determined by the enzymaticmethod (Donis-Keller, H., (1980) Nucleic Acids Res. 8:3133-3142). Somenucleotides could not be assigned due to presence of a band in theuntreated (control) RNA sample. G^(*), labeled GTP joined to the RNAduring the reaction.

Methods: L-19 IVS RNA was synthesized by methods similar to thosedescribed previously (Zaug, A. J., and Cech, T. R., (1986) Science231:470-475), except a different RNA polymerase-template system wasused. Plasmid pT7-TT1A3 (which contains a T7 RNA polymerase promoter, a42 bp 5' exon, the entire 413 bp IVS, and an 82 bp 3' exon) was cleavedwith Eco RI and transcribed with bacteriophage T7 RNA polymerase(Davanloo, P., et al. (1984) Proc. Nat'l. Acad. Sci. U.S.A. 81:2035-2039). The transcripts were incubated further under self-splicing,cyclization, and site-specific hydrolysis conditions to produce L-19 IVSRNA (Zaug, A. J., et al. (1984) Science 224:574-578; Zaug, A. J. andCech, T. R., (1986) Science 231:470-475). The 3'-terminal guanosine wasthen removed by periodate oxidation and beta-elimination (Winter, G., etal. (1978) Nucleic Acids Res. 5, 3129-3139) to yield L-19 IVS-beta RNA.Substrate RNAs were produced by T7 RNA polymerase transcription ofBamHI-cleaved pAK105 (Mount, S. M., et al. (1983) Cell 33:509-518),XmnI- or ScaI-cleaved pT7-1 (purchased from U.S. Biochemical Corp.), andSnaBI-cleaved pDW27, which encodes Ml RNA (obtained from D. Wahl and N.Pace). Substrate RNAs were incubated with L-19 IVS-beta RNA in 5 mMMgCl₂, 10 mM NaCl, 50 mM Tris-HCl, pH 7.5 at 50° C.; in addition, GTPwas present at the concentration indicated. Reactions were stopped bythe addition of EDTA to a final concentration of 25 mM. Products wereanalyzed by electrophoresis in 4% polyacrylamide, 8M urea gels andsubjected to fluorography (a) or autoradiography (b).

FIG. 3 Three different ribozymes can distinguish between sequences thatdiffer by only one single-base change within the recognition element a,Synthesis of defined oligoribonucleotide substrates by the method ofLowary et al. (Lowary, P., et al. NATO ASI Series A, vol. 110, 69-761986). DNA was transcribed with T7 RNA polymerase in the presence of[alpha-³² P]ATP to produce oligoribonucleotides labeled with ³² P in thepositions indicated (*). b, Proposed interactions between the threeoligoribonucleotide substrates (top strands, boldface letters) and theactive sites of the matched ribozymes (bottom strands). Arrows designatesites of cleavage and guanosine addition c, Cleavage of 3oligoribonucleotide substrates by wild-type and variant L-19 IVS RNAs,assayed by 20% polyacrylamide, 7M urea gel electrophoresis. (-),untreated substrate. In other pairs of lanes, 1.0 uM ³² p-labeledsubstrate was incubated with 0.125M ribozyme for 15 min (left lane) or60 min (right lane) at 50° C. in 10 mM MgCl₂, 10 mM NaCl, 50 mMTris-HCl, pH 7.5, 0.5 mM GTP (unlabeled), 2.5M urea. Because the ³² P isconfined to the nucleotides downstream from the cleavage site, only thesmaller of the products is apparent. The identity of the GA₅ product wasconfirmed by treatment of the substrate and reaction mixtures with RNaseT₂ and monitoring the transfer of ³² P to Gp. The band migrating aboveGA₅ in lanes 2 and 3 was not produced consistently and has not beenidentified.

Methods: Substrates were prepared by transcription ofdeoxyoligonucleotides synthesized on an Applied Biosystems DNASynthesizer. The same promoter top strand was used in eachtranscription. The bottom strand, which contains promoter and templatesequences, was varied to obtain the different RNA substrates. The DNAwas transcribed with purified phage T7 RNA polymerase (Davanloo, P., etal. (1984) Proc. Nat'l. Acad. Sci. U.S.A. 81:2035-2039) as described(Lowary, P., et al. NATO ASI Series, vol. 110, as above). Variantribozymes are described by Been and Cech (Been, M. D., et al. (1986)Cell, 47:207-216). The 24C ribozyme was similarly prepared fromtranscripts of pBG/-3G:24C. The variant ribozymes were not subjected tobeta-elimination to remove their 3'-terminal G.

FIG. 4 Kinetic analysis of the RNA endoribonuclease reaction. a, Theoligoribonucleotide substrate (2.5 uM) was incubated with wild-type L-19IVS-beta RNA (0.2 uM) as in FIG. 3, except that urea was omitted. b,Kinetics of cleavage of GGCCCUCUA₅ as a function of GTP concentration.RNA substrate concentration was kept constant at 2.5 uM. c, Kinetics ofcleavage as a function of RNA substrate concentration, with GTPconcentration kept constant at 0.5 mM.

Methods: Products were separated by polyacrylamide gel electrophoresis.With the autoradiogram as a guide, each gel was cut into strips, and theradioactivity in the unreacted substrate and the GA₅ product wasdetermined by liquid scintillation counting. The initial velocity ofcleavage (Vo) was determined from a semilogarithmic plot of the fractionof reaction as a function of time. 1/Vo was then plotted as a functionof inverse substrate concentration; the graphs are linear least-squaresfits to the data points.

FIG. 5 The L-19 IVS RNA catalyzes the cleavage and rejoining ofoligoribonucleotide substrates; (A) 10 uM pC₅ and (B) 10 uM d-pC₅, bothwith 1.6 uM L-19 IVS RNA; (C) 45 uM pC₅ in the absence of L-19 IVS RNA;(D) 45 uM pU⁶ with 1.6 uM L-19 IVS RNA; (E) 10 uM pC₅, (F) 50 uM pC₅ and(G) 100 uM pC₅, all with 1.6 uM L-19 IVS RNA. Oligonucleotides were5'-end labeled by treatment with [gamma-³² P]ATP and polynucleotidekinase; they were diluted with unlabeled oligonucleotide of the samesequence to keep the amount of radioactivity per reaction constant TheL-19 IVS RNA was synthesized by transcription and splicing in vitro.Supercoiled pSPTT1A3 DNA (Price, J. V., et al. (1985) Science 228:719)was cut with Eco RI and then transcribed with SP6 RNA polymerase(Butler, E. T., et al. (1982) J. Biol. Chem. 257:5772; Melton, D. A., etal. (1984) Nucleic Acids Res. 12:7035) for 2 hours at 37° C. in asolution of nucleoside triphosphates (0.5 mM each), 6 mM MgCl₂, 4 mMspermidine, 10 mM dithiothreitol, 40 mM tris-HCl, pH 7.5, with 100 unitsof SP6 RNA polymerase per microgram of plasmid DNA. Then NaCl was addedto a final concentration of 240 mM and incubation was continued at 37°C. for 30 minutes to promote excision and cyclization of the IVS RNA.Nucleic acids were precipitated with three volumes of ethanol andredissolved in 50 mM CHES, pH 9.0; MgCl₂ was added to a finalconcentration of 20 mM, and the solution was incubated at 42° C. for 1hour to promote site-specific hydrolysis of the circular IVS RNA to giveL-19 IVS RNA (Zaug, A. J., et al., Science 224, 574 (1984). The reactionwas stopped by the addition of EDTA to 25 mM. The L-19 IVS RNA waspurified by preparative gel electrophoresis and Sephadex G-50chromatography. Labeled oligonucleotides were incubated with unlabeledL-19 IVS RNA at 42° C. in 20 mM MgCl₂, 50 mM tris, pH 7.5, for 0, 1, 2,5, 10, 30, and 60 minutes. Reactions were stopped by the addition ofEDTA to a final concentration of 25 mM. Products were analyzed byelectrophoresis in a 20 percent polyacrylamide, 7M urea gel,autoradiograms of which are shown.

FIG. 6 Kinetics of conversion of pC₅ to larger and smalleroligonucleotdides with 1.6 uM L-19 IVS RNA. Products were separated bypolyacrylamide gel electrophoresis. With the autoradiogram as a guide,the gel was cut into strips and the radioactivity in each RNA specieswas determined by liquid scintillation counting. The amount of reactionat each time was taken as the radioactivity in pC₃ +pC₄ +pC₆ +pC₇ + . .. divided by the total radioactivity in the lane. The initial velocityof product formation, V_(o), was determined from a semilogarithmic plotof the fraction of reaction as a function of time. V_(o) was thenplotted as a function of substrate concentration; the line is aleast-squares fit to the Michaelis-Menten equation. The resultingkinetic parameters are K_(m) =42 uM, V_(max) =2.8 uM min⁻¹, and k_(cat)=1.7 min⁻¹. The kinetic parameters for the first and second steps in thereaction have not yet been determined separately.

FIG. 7 Formation and resolution of the covalent enzyme-substrateintermediate. (A) To form the covalent L-19 IVS RNA-substrateintermediate, 8.5 nM C₅ p^(*) was treated with 0.16 uM L-19 IVS RNAunder standard reaction conditions for 0 to 60 minutes. (B) p^(*) C₅(0.01 uM) was reacted with 0.16 uM L-19 IVS RNA. Cleavage occurrednormally, but there was very little rejoining. (C) Labeled covalentintermediate was prepared as in (A) (60 minutes) and purified byelectrophoresis in a 4 percent polyacrylamide, 8M urea gel. It was thenincubated with 10 uM unlabeled C₅ under standard reaction conditions for0 to 60 minutes. The product designated C₆ comigrated with labeled C₆marker (not shown). (D) Isolated covalent intermediate as in (C) wasincubated under site-specific hydrolysis conditions (20 mM MgCl₂, 50 mMCHES, pH 9.0) at 42° C. for 0 to 60 minutes. Positions of labeled mono-and dinucleotide markers are indicated. In the 10- and 30-minute lanesof (A) and the 10-, 30-, and 60-minute lanes of (C), band compression(reduced difference in electrophoretic mobility) is seen between C₆ andC₇ and to a lesser extent between C₇ and C₈. This is due to the absenceof a 5' phosphate. Thus, the charge-to-mass ratio is increasing withchain length, whereas with 5'-phosphorylated oligonucleotides thecharge-to-mass ratio is independent of chain length. When such productswere phosphorylated by treatment with polynucleotide kinase and ATP, thedistribution was converted to the normal spacing as in FIG. 1 [Zaug, A.,et al. (unpublished data)].

FIG. 8 Model for the enzymatic mechanism of the L-19 IVS RNA. The RNAcatalyzes cleavage and rejoining of oligo(C) by the pathway 1 - 2 - 3 -4 - 1. The L-19 IVS RNA enzyme (1) is shown with the oligopyrimidinebinding site (RRRRRR, six purines) near its 5' end and G⁴¹⁴ with a free3'-hydroxyl group at its 3' end. The complex folded core structure ofthe molecule (Davies, R. W., et al., (1982) Nature (London) 300:719;Waring, R. B., et al. (1983) J. Mol. Biol. 167:595; Michel, F., et al.(1983) EMBO J. 2:33; Cech, T. R., et al. (1983) Proc. Nat'l. Acad. Sci.U.S.A. 80:3903; Inoue, T., et al. (1985) ibid 82:648) is simplyrepresented by a curved line. The enzyme binds its substrate (C₅) byWatson-Crick base-pairing to form the noncovalent enzyme-substratecomplex (2). Nucleophilic attack by G⁴¹⁴ leads to formation of thecovalent intermediate (3). With the pentanucleotide C₅ as substrate, thecovalent intermediate is usually loaded with a single nucleotide, asshown; with substrates of longer chain length, an oligonucleotide can beattached to the 3' end of G⁴¹⁴. If C₅ binds to the intermediate (3) inthe manner shown in (4), transesterification can occur to give the newproduct C₆ and regenerate the enzyme (1). Note that all four reactionsin this pathway are reversible. When acting as a ribonuclease, the L-19IVS RNA follows the pathway 1 - 2 - 3 - 1. The covalent intermediate (3)undergoes hydrolysis, releasing the nucleotide or oligonucleotideattached to its 3' end (in this case pC) and regenerating the enzyme(1).

FIG. 9. Competitive inhibition of the pC₅ reaction by d-C₅. (A) 5 uMp^(*) C₅, shown unreacted in lane O, was incubated with 0.16 uM L-19 IVSRNA under standard reaction conditions. Reactions were done in theabsence of d-C₅ or in the presence of 50 uM, 500 uM, or 1000 uM d-C₅ asindicated. (B) Lineweaver-Burk plots of the rate of conversion of pC₅ topC₄ +pC₃ in the presence of (o) 0 uM, (open square) 50 uM, (opentriangle) 150 uM, (closed circle) 300 uM, or (closed square) 500 uMunlabeled d-C₅. The analysis was limited to the smaller products becausetheir production is affected only by the first transesterificationreaction (FIG. 8). Although d-C₅ is inactive in the firsttransesterification reaction, it has some activity as a substrate in thesecond transesterification reaction (Zaug, A., et al. unpublished data)and therefore could affect the production of chains of length greaterthan 5 . (C K_(m) /V_(max), determined from the slopes of the lines in(B), is plotted against the inhibitor concentration. The x-interceptgives the negative of K_(i) ; K_(i) =260 uM.

FIG. 10 Relation of reactions catalyzed by the L-19 IVS RNA toself-splicing and the related IVS RNA-mediated reactions. Formation ofthe covalent enzyme-substrate intermediate (A) is analogous to IVS RNAautocyclization (B). Resolution of the enzyme-substrate intermediate (C)is analogous to exon ligation (D) or the reversal of cyclization(Sullivan, F. X. and Cech, T. R. (1985) Cell 42:639). Hydrolysis of theenzyme-substrate intermediate (E) is analogous to site-specifichydrolysis of the circular IVS RNA (F) or the pre-rRNA (Inoue, T., etal. (1986) J. Mol. Biol. 189:143-165).

FIG. 11 The L-19 IVS RNA catalyzes the dephosphorylation ofoligo(cytidylic acid) 3'-phosphate. (A) L-19 IVS RNA (0.16 uM) wasincubated with p^(*) C₅ (10 uM), p^(*) A₆ (10 uM), C₅ p^(*) Cp(about 2nM), and A₆ p^(*) Cp (about 3 nM) in 20 mM MgCl₂ and 50 mM Tris-HCl, pH7.5, at 42° C. for the times indicated. Reaction products were separatedby electrophoresis in a 20% polyacrylamide-7M urea sequencing gel, anautoradiogram of which is shown. (B) L-19 IVS RNA (0.2 uM) was incubatedwith C₅ p^(*) (about 2 nM) as above. The phosphoenzyme E-p^(*) is theL-19 IVS RNA with a 3'-terminal phosphate monoester. Gel electrophoresisand autoradiography as in (A). Only a portion of the 5-min sample wasloaded on the gel.

FIG. 12 Effect of pH on the phospho transfer and nucleotidyl transferreactions. (A) Lane 1, untreated C₅ p^(*) Cp; lanes 2-11, C₅ p^(*) Cp(15 nM) incubated with excess L-19 IVS RNA (500 nM) in 20 mM MgCl₂ and50 mM buffer (NaOAc for pH 4.0 and 5.0, Tris-HCl for pH 7.5, CHES for pH9.0 and 10.0); lane 12, C₅ p^(*) Cp treated with calf intestinalphosphatase to provide a marker for C₅ p^(*) C--OH; lane 13, untreatedp^(*) C₅ ; lanes 14-23, p^(*) C₅ (15 nM) incubated with excess L-19 IVSRNA (500 nM) as in lanes 2-11. Reactions proceeded at 42° C. for theindicated times, after which they were stopped by he addition of anequal volume of urea sample buffer containing 50 mM EDTA. (B) C₅ p^(*)Cp (about 2 nM) was incubated with L-19 IVS RNA (0.2 uM) at pH 5.0 forthe times indicated. (C) Data similar to those shown in (B) except with15 nM C₅ p^(*) Cp were quantitated by liquid scintillation counting ofthe sliced gel. Semilogarithmic plots, which were linear for the firstthree or four time points, were used to determine t_(1/2). The observedfirst-order rate constant (k_(obsd)) was calculated as (1n 2)/t₁₇₈.NaOAc buffer was used for pH 4.0 and 5.0, MES for pH 5.5 and 6.0, andTris-HCl for pH 7 (estimate based on a single point that showed about50% reaction).

FIG. 13. Phosphorylation of the enzyme is reversible. L-19 IVS RNA (0.2uM) was phosphorylated by incubation with 2.5 uM unlabeled C₅ p for minat 42° C. at pH 5.0. A trace amount of (A) C₅ p^(*) (1.2 nM) or (B)p^(*)C₅ (20 nM) was then added to the unlabeled E-p, and incubation wascontinued for the times shown.

FIG. 14. The L-19 IVS RNA acts catalytically as a phosphotransferase.(A) Decreasing concentrations of C₅ p^(*) were incubated for 30 min with0.32 uM L-19 IVS RNA and 100 uM unlabeled UCU-OH. The specificradioactivity of C₅ p^(*) was adjusted by addition of unlabeled C₅ p^(*)to keep the amount of radioactivity constant among samples. The smallamount of intermediate band seen in some reactions is presumed to beUCUCp formed by attack of UCU on an E-pCp covalent intermediate. (-lane)C₅ p^(*) prior to incubation. (B) C₅ p^(*) (2.5 uM) incubated with 0.16uM L-19 IVS RNA and 200 uM unlabeled UCU-OH. (C) Quantitation of datashown in (B), including labeled E-p, which ran near the top of the gel.In all cases, incubation was in 20 mM MgCl₂ and 50 mM MES, pH 6.0, at42° C.

FIG. 15. Single active site model for the activity of the L-19 IVS RNAon phosphate diester and phosphate monoester substrates. (A) Reversiblenucleotidylation of the L-19 IVS RNA, proposed to be the key step in thepoly(C) polymerase reaction (Zaug & Cech, (1986) Science (Wash. D.C.231:470-475. (B) Reversible phosphorylation of the L-19 IVS RNA, whichallows the enzyme to act as a phosphotransferase. In both cases, theoligo(C) substrate base pairs to the oligo(Pyrimidine) binding site (sixR's) to form a noncovalent complex. The binding site is nucleotides22-27 of the IVS RNA and has the sequence GGAGGG (M. D. Been and T. R.Cech, (1986) Cell 47:206-216). Nucleophilic attack by the 3'-hydroxyl ofthe active site guanosine, G414 leads to formation of E-pC (A) or E-p(B) and the release of C₅. The complex folded core structure of the IVSis depicted as a simple curved line.

FIG. 16 Plasmid pBGST7 contains a fragment with the IVS inserted intothe multicloning site of a small pUC18 derivative which in turn containsa phage T7 promoter. The double line represents the plasmid DNA with theIVS. The relative positions of the promoters are indicated. Thepositions of the EcoRI and Hind III site are indicated by thearrowheads. The upper line represents the in vitro transcript made frompurified plasmid DNA with phage T7 RNA polymerase. The numbers above itrefer to the length, in nucleotides, of the exons and IVS. The lowerline represents the in vivo transcript of the 5' end of lacZ'. The IVS(heavy line) contains stop codons in all three reading frames, so afunctional alpha fragment can only be produced if the IVS is excised andthe exons are joined by splicing in E. coli.

DESCRIPTION

The Tetrahymena rRNA intervening sequence (IVS) is a catalytic RNAmolecule or ribozyme. It mediates RNA self-splicing, accomplishing itsown excision from the large ribosomal RNA precursor, and subsequentlyconverts itself to a circular form (Kruger, K., et al. (1982) Cell31:147-157; Zaug, A. J., et al. (1983) Nature 301:578-583). In thesereactions, the splice sites and cyclization sites can be viewed asintramolecular substrates for an activity that resides within the IVSRNA (Zaug, A. J., et al. (1984) Science 224:574-578). This is not a trueenzymatic reaction however since the RNA is not regenerated in itsoriginal form at the end of the self-splicing reaction. The IVS RNA whenin its linear form is referred to as L IVS RNA.

This view has been validated by studies of the L-19 IVS RNA, a linearform of the IVS which is missing the first 19 nucleotides. Because itlacks the cyclization sites, the L-19 IVS RNA cannot undergointramolecular reactions (Zaug, A. J., et al. (1984) Science224:574-578). It still retains activity, however, and can catalyzecleavage-ligation reactions on other RNA molecules (Zaug, A. J. andCech, T. R. (1986) Science 231:470-475). When provided witholigo(cytidylic acid) as a substrate, the L-19 IVS RNA acts as an enzymewith nucleotidyltransferase [poly(C) polymerase] and phosphodiesterase(ribonuclease) activities (Zaug, A. J. and Cech, T. R. (1986) Science231:470-475). With 3'-phosphorylated oligo(C) substrates, the sameribozyme acts as a phosphotransferase and an acid phosphatase (Zaug, A.J. and Cech, T. R. (1986) Biochemistry 25:4478-4482). A key mechanisticfeature of all four of these reactions is the formation of a covalentenzyme-substrate intermediate in which a nucleotide or phosphate isesterified through the 3'-0 of G⁴¹⁴, the 3' terminal guanosine of theIVS RNA. In addition, we describe herein a fifth enzymatic activityconcerning the endoribonuclease activity of the L-19 IVS RNA on otherRNA molecules.

Following self-splicing of the Tetrahymena rRNA precursor, the excisedIVS RNA (Abbreviations: IVS, intervening sequence or intron: L-19 IVSRNA (read "L minus 19"), a 395-nt RNA missing the first 19 nt of the LIVS RNA (the direct product of pre-ribosomal RNA splicing); p^(*),³² Pwithin an oligonucleotide, that is, C₅ p^(*) C is CpCpCpCpC³² pC andp^(*) C₅ is ³² pCpCpCpCpC; d-C₅, deoxyC₅) undergoes a series ofRNA-mediated cyclization and site-specific hydrolysis reactions. Thefinal product, the L-19 IVS RNA is a linear molecule that does not havethe first 19 nucleotides of the original excised IVS RNA (Zaug, A. J.,et al., Science 224:574 (1984). We interpreted the lack of furtherreaction of the L-19 species as an indication that all potentialreaction sites on the molecule that could reach its active site (thatis, intramolecular substrates) had been consumed; and we argued that theactivity was probably unperturbed (Zaug, A. J., et al., Science 224:574(1984) (L IVS RNA is linear IVS RNA). We have now tested this by addingoligonucleotide substrates to the L-19 IVS RNA. We find that each IVSRNA molecule can catalyze the cleavage and rejoining of manyoligonucleotides. Thus, the L-19 IVS RNA is a true enzyme. Although theenzyme can act on RNA molecules of large size and complex sequence, wehave found that studies with simple oligoribonucleotides like pC₅(pentacytidylic acid) have been most valuable in revealing the minimumsubstrate requirements and reaction mechanism of this enzyme.

Nucleotidyltransferase or RNA Polymerase Activity

When the shortened form of the self-splicing ribosomal RNA (rRNA)intervening sequence of Tetrahymena thermophila acts as a nucleotidyltransferase, it catalyzes the cleavage and rejoining of oligonucleotidesubstrates in a sequence-dependent manner with K_(m) =42 uM and k_(cat)=2 min⁻¹. The reaction mechanism resembles that of rRNA precursorself-splicing. With pentacytidylic acid as the substrate, successivecleavage and rejoining reactions lead to the synthesis of polycytidylicacid. When the active site is changed from the natural nucleotidesequence GGAGGG to the sequence GAAAAG, oligouridylic acid ispolymerized to polyuridylic acid [Been and Cech, Cell 47:207 (1986)].Thus, the RNA molecule can act as an RNA polymerase, differing from theprotein enzyme in that it uses an internal rather than an externaltemplate. Thus various heteropolymers would be constructed by varientRNA enzyme forms. This predicts the formation for example of messengerRNA molecules for particular peptides or proteins. This messenger couldbe synthesized with or without introns. At about pH 9, the same RNAenzyme has activity as a sequence-specific ribonuclease.

With C₅ as substrate, the L-19 IVS RNA makes poly(C) with chain lengthsof 30 nucleotides and longer, acting as an RNA polymerase or nucleotidyltransferase. Thus longer oligonucleotides (polynucleotides) can beformed from short oligonucleotide starting material The number of P--Obonds is unchanged in the process. In the synthesis of poly(C) on apoly(dG) template by RNA polymerase, one CTP is cleaved for each residuepolymerized. Thus, the RNA polymerase reaction is also conservative withrespect to the number of P--O bonds in the system. The L-19 IVS RNA cantherefore be considered to be a poly(C) polymerase that uses C₄ pCinstead of pppC as a substrate. It incorporates pC units at the 3' endof the growing chain and releases C₄ ; the C₄ is analogous to thepyrophosphate released by RNA polymerase. Synthesis is directed by atemplate, but the template is internal to the RNA enzyme. It may bepossible to physically separate the template portion from the catalyticportion of the RNA enzyme with retention of activity. If so, the RNAenzyme could conceivably act as a primordial RNA replicase, catalyzingboth its own replication and that of other RNA molecules (T. R. Cech,(1986) Proc. Nat'l. Acad. Sci. USA 83:4360-4363.

The L-19 IVS RNA catalyzes the cleavage-ligation of pC₅ with K_(m) =42uM, k_(cat) =2 min⁻¹, and k_(cat) /K_(m=) 1×10³ sec⁻¹ M⁻¹. The K_(m) istypical of that of protein enzymes. The k_(cat) and k_(cat) /K_(m) arelower than those of many protein enzymes. However, k_(cat) within therange of values for proteins that recognize specific nucleic acidsequences and catalyze chain cleavage or initiation of polymerization.For example, Eco RI restriction endonuclease cleaves its recognitionsequence in various DNA substrates, including a specific 8-bp DNAfragment, with k_(cat) =1 min⁻¹ to 18 min⁻¹ (Greene, P. J., et al.(1975) J. Mol. Biol. 99:237; Modrich, et al. (1976) J. Biol. Chem251:5866; Wells, R. D., et al. (1981) Enzymes 14:157; Brennan, M. B., etal. in preparation; and Terry, B., et al. in preparation). The k_(cat)is also similar to that of the RNA enzyme ribonuclease P, which cleavesthe precursor to tRNA with k_(cat) =2 min⁻¹ (Guerrier-Takada, C., et al.(1983) Cell 35:849; Marsh, T. L., et al. in Sequence Specificity inTranscription and Translation, R. Calendar and L. Gold Eds., UCLASymposium on Molecular and Cellular Biology (Plenum, New York, inpress).

Another way to gauge the catalytic effectiveness of the L-19 IVS RNA isto compare the rate of the catalyzed reaction to the basal chemicalrate. A transesterification reaction between two free oligonucleotideshas never been observed, and hence the uncatalyzed rate is unknown. Onthe other hand, the rate of hydrolysis of simple phosphate diesters hasbeen studied (Kumamoto, J. et al. (1956) J. Am. Chem Soc. 78:4858; P. C.Haake et al. ibid (1961) 83:1102; Kirby, A. J., et al. (1970) J. ChemSoc. Ser. B., p. 1165; Bunton, C. A., et al. (1969) J. Org. Chem.34:767)). The second-order rate constant for alkaline hydrolysis of thelabile phosphodiester bond in the circular IVS RNA (Zaug, A. J., et al.(1985) Biochemistry 24:6211) is 12 orders of magnitude higher than thatof dimethyl phosphate (Kumamoto, J., et al (1956) Supra) and ten ordersof magnitude higher than that expected for a normal phosphodiester bondin RNA (The rate of nucleophilic attack by hydroxide ion on phosphateesters is sensitive to the pK_(a) of the conjugate acid of the leavinggroup. A phosphate in RNA should be more reactive than dimethylphosphate, because pK_(a) =12.5 for a nucleoside ribose and pK_(a) =15.5for methanol [values at 25° C. from P.O.P. T'so, Basic Principles inNucleic Acid Chemistry (Academic Press, New York, (1974), vol. I, pp.462-463 and P. Ballinger and F. A. Long, J. Am. Chem. Soc. 82:795(1960), respectively). On the basis of the kinetic data available forthe alkaline hydrolysis of phosphate diesters (Kumamoto, J , et al.(1956) J. Am. Chem Soc. 78:4858; Haake, P. C. (1961) et al. ibid83:1102; Kirby, A. J., et al (1970) J. Chem Soc. Ser. B., p. 1165;Bunton, C. A., et al. (1969) J. Org. Chem 34:767), the slope of a graphof the logarithm of the rate constant for hydrolysis as a function ofpK_(a) can be roughly estimated as 0.6. Thus, RNA is expected to be morereactive than dimethyl phosphate by a factor of 10⁰.6 (15.5-12.5)=10¹.8. The estimate for RNA pertains to direct attack by OH⁻ on thephosphate, resulting in 3'-hydroxyl and 5'-phosphate termini. Cleavageof RNA by OH⁻ -catalyzed transphosphorylation, producing a 2',3'-cyclicphosphate, is a much more rapid (intramolecular) reaction but is notrelevant to the reactions of the L-19 IVS RNA). On the basis of the dataof FIG. 7D, the covalent enzyme-substrate complex undergoes hydrolysisat approximately the same rate as the equivalent bond in the circularIVS RNA. Thus, we estimate that the L-19 IVS RNA in its ribonucleasemode enhances the rate of hydrolysis of its substrate about 10¹⁰ times.

The RNA moiety of ribonuclease P, the enzyme responsible for cleavingtransfer RNA (tRNA) precursors to generate the mature 5' end of thetRNA, is an example of an RNA enzyme molecule. (Guerrier-Takada, C., etal., (1983) Cell 35:849; Guerrier-Takada, C., et al. (1984) Science223:285; Marsh, T. L., et al. in Sequence Specificity in Transcriptionand Translation, R. Calendar and L. Gold Eds., UCLA Symposium onMolecular and Cellular Biology (Plenum, New York, in press); Marsh, T.L., et al (1985) Science 229:79). However, this enzyme catalyzes only aspecific tRNA reaction without general RNA activity. The specificity issuch that a variety of single base changes in the t-RNA portion of thepre-tRNA substrate prevent the enzyme from cleaving the substrate.

Dephosphorylation Activity

We have also found that the same enzyme has activity toward phosphatemonoesters. The 3'-phosphate of C₅ p or C₆ p is transferred to the3'-terminal guanosine of the enzyme. The pH dependence of the reaction(optimum at pH 5) indicates that the enzyme has activity toward thedianion and much greater activity toward the monoanion form of the3'-phosphate of the substrate. Phosphorylation of the enzyme isreversible by C₅ --OH and other oligo(pyrimidines) such as UCU-OH. Thus,the RNA enzyme acts as a phosphotransferase, transferring the3'-terminal phosphate of C₅ p to UCU-OH with multiple turnover At pH 4and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganicphosphate. Thus, the enzyme has acid phosphatase activity. These are thetwo aspects of its dephosphorylase activity. The RNA enzymedephosphorylates oligonucleotide substrates with high sequencespecificity, which distinguishes it from known protein enzymes.

The L-19 IVS RNA has transphosphorylation activity toward3'-phosphorylated oligo(C) substrates. The properties of thetransphosphorylation reaction indicate that it is taking place in thesame active site as the poly(C) polymerase and ribonuclease reactions(FIG. 15). The properties include the specificity of the reactions foroligo(C) substrates, the production of oligo(C) products with3'-hydroxyl termini, and the formation of similar covalentenzyme-substrate complexes. The presumptive intermediate is aphosphoenzyme, E-p, in the case of the phosphotransferase reaction and anucleotidyl enzyme, E-pC or E-(pC)_(n), in the case of the poly(C)polymerase and ribonuclease reactions (Zaug & Cech, (1986) Science(Wash., D.C. 231:470-475). In both cases the presumptive covalentintermediate involves a phosphate ester linkage through the 3'-0 of G414of the L-19 IVS RNA (Zaug and Cech, unpublished results).

The transphosphorylation reaction is readily reversible. The phosphatecan be transferred from the enzyme to an acceptor with a 3'-hydroxylgroup, such as C₅ or UCU. With C₅ p and UCU as cosubstrates, the L-19IVS RNA can catalyze the reaction C₅ p+UCU-OH --C₅ --OH+UCUp. Theproposed pathway is ##STR1## Thus, the L-19 IVS RNA hastransphosphorylation activity resembling that of Escherichia colialkaline phosphatase (Reid & Wilson, (1971) Enzymes (3rd Ed.)4:373-415); Coleman & Gettins, (1983) Adv. Enzymol. Relat. Area Mol.Biol., 55:381), acid phosphatase, and a variety of otherphosphotransferases that form covalent enzyme-substrate intermediates(Knowles, (1980) Ann. Rev. Biochem 49:877). In addition, the L-19 IVSRNA phosphoenzyme can transfer its phosphate to water at pH 4 and 5,indicating it has acid phosphatase activity.

As the pH is lowered from 7.5 to 5.0, the rate of thetransphosphorylation reaction increases substantially. In this same pHrange, the 3'-phosphate of C₅ p is converted to a monoanion [pK_(a)approximately 6.0, based on the value for cytidine 3'-phosphate fromTs'o [(1974) Basic Principles in Nucleic Acid Chemistry vol. 1, pp. 462Academic, N.Y.]. Protonation of a phosphate monoester makes it possiblefor it to react like a diester (Benkovic & Schray, (1973) Enzymes (3rdEd.) 8:235). Thus, it seems reasonable that an enzyme known to reactwith diesters could use the same mechanism to react with monoestermonoanions. The acidic pH requirement for hydrolysis of thephosphoenzyme can be similarly explained if the reaction occurs byattack of water on the phosphate monoester monoanion. The pHindependence of transphosphorylation between pH 7.5 and pH 9.0 stronglysuggests that the monoester dianion is also reactive, albeit at a rateless than 5% that of the monoester monoanion. The reactivity of themonoester dianion is surprising and perhaps indicates that the enzymeprovides electrophilic assistance to the departure of the leaving groupwith a proton or metal ion.

At alkaline pH the phosphodiester bond following G414 is labile in thecircular IVS RNA (Zaug et al., 1984 Science (Wash., D.C. 224:574), inthe pre-rRNA (Inoue et al., 1986, J. Mol. Biol 189:143), and in E-pC(Zaug, A. J., and Cech, T., 1986, Science (Wash., D.C.) 231:470-475),whereas the phosphomonoester bond following G⁴¹⁴ is stable in E-p.Specific hydrolysis of the phosphodiester bonds involves attack ofhydroxide ion (Zaug, A. J. et al., (1985) Biochemistry 24:6211). It isnot surprising that attack of hydroxide ion on the phosphate monoesterdianion of E-p might be prohibited due to electrostatic repulsion (Kirby& Younas, (1970) J. Chem. Soc. B, 1165).

At pH 5 the phosphoenzyme undergoes very slow hydrolysis but readilytransfers its phospho group to C₅ --OH. The rate of the hydrolysisreaction is 2-3 orders of magnitude slower than that of the phosphotransfer reaction, even though H₂ O is present at 55M and theoligonucleotide at less than 1 uM. Thus, C₅ --OH is a better acceptorthan H₂ O by a factor exceeding 10¹⁰. [Such a large factor is notunusual for phosphotransferases; for example, Ray et al. (1976)Biochemistry 15:4006 report that phosphoglucomutase transfers aphosphate to the C-6 hydroxyl of glucose 1-phosphate at a rate 3×10¹⁰times greater than that of transfer to H₂ O.] The difference in rate ismuch too large be explained by the greater nucleophilicity of the3'-hydroxyl of C₅ --OH than H₂ O, which could perhaps account for afactor of 10 (Lohrmann & Orgel (1978) Tetrahedron 34:853; Kirby &Varvoglis, (1967) J. Am. Chem. Soc. 89:415-423). Most of the enzyme toutilize the binding reflects the ability of the enzyme to utilize thebinding energy from its interaction with non-reacting portions of C₅--OH (Jencks, W. P., 1975, Adv. Enzymol. Relat. Areas Md. Biol. 43:219).For example, specific binding interactions could precisely position the3'-hydroxyl of C₅ --OH for displacement of the phosphate from theenzyme, but would not be available to facilitate the addition of water.Furthermore, the catalytic apparatus may not be fully assembled untilthe acceptor oligonucleotide is in place and water is absent (Koshland,(1963) Cold Spring Harbor Symp. Quant. Biol. 28:473; Knowles, (1980)Ann. Rev. Biochem. 49:877).

We are only beginning to understand how the L-19 IVS RNA catalyzesphospho transfer. The overall transfer reaction is undoubtedlyfacilitated by the formation of a covalent bond between the enzyme andthe phosphate of the substrate. Such covalent catalysis is common inenzyme-catalyzed group transfer reactions (Jencks, (1969) Catalysis inChemistry and Enzymology McGraw-Hill, New York; Walsh, (1979) EnzymaticReaction Mechanisms W. H. Freeman, San Francisco). Binding sites withinthe IVS RNA for the oligo(pyrimidine) substrate (Zaug & Cech, (1986)Science (Wash., D.C. 231:470-475) and for the nucleophilic G residue atits own 3' end (N. K. Tanner and T. R. Cech, unpublished results)contribute to the catalysis of the transfer reactions. These bindinginteractions presumably place the 3'-hydroxyl group of G414 in anoptimal orientation for nucleophilic attack on the terminal phosphate ofC₅ p (FIG. 15B) or on an internal phosphate of C₅ --OH (FIG. 15A). Wesuspect that catalysis might also involve a specific role for Mg²⁺[Steffens et al., (1973) J. Am. Chem. Soc. 95:936 and (1975)Biochemistry 14:2431; Anderson et al., (1977) J. Am. Chem. Soc. 99:2652;see also Zaug et al. (1985) Biochemistry 24:6211 and Guerrier-Takada etal. (1986) Biochemistry 25:1509] and general acid-base catalysis [seeCech and Bass (1986) Ann. Rev. Biochem. 55:599-629], but we have nodirect evidence for such mechanisms. Applicants are not specificallybound by only those mechanisms discussed herein.

One unanswered question concerns the extremely low extent of nucleotidyltransfer with the C₅ p substrate at neutral pH. Since C₅ --OH is readilyattacked at the phosphate following the fourth C to produce E-pC, why isC₅ p not attacked at the equivalent phosphate to produce E-pCp? Perhapsthe terminal phosphate of C₅ p is coordinated to Mg(II) or serves as ahydrogen bond acceptor, resulting in a preferred mode of bindingdifferent from that of C₅ --OH.

Finding an enzyme that has both phosphodiesterase andphosphomonoesterase activity is unusual but not unprecedented.Exonuclease III (Richardson & Kornberg, (1964) J. Biol. Chem.239:242-250), P1 nuclease, and mung bean nuclease (Shishido & Ando, 1982in Nucleases (Linn, S. M. & Roberts, R. J. Eds) pp. 155-185, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) all have 3'-phosphataseactivity.

The L-19 IVS RNA is unique among known enzymes in its ability to remove3'-phosphates from RNA with high substrate specificity. E. coli andmammalian alkaline phosphatases are nonspecific. These enzymes remove5'-, 3'-, and 2'-phosphates from RNA with little regard for the basesequence of the molecule (Garen & Levinthal, (1960) Biochem. Biophys.Acta. 38:470; Harkness, (1968) Arch. Biochem. Biophys. 126:513).Polynucleotide kinase has 3'-phosphatase, 2'-phosphatase, and cyclic2',3'-phosphatase activity (Cameron & Uhlenbeck, (1977) Biochemistry16:5120; Weber, (1985) Ph.D. Thesis, University of Illinois). Substratesas diverse as U₅ p, A₆ Cp, and pCp are readily dephosphorylated, andwhere careful kinetic measurements have been made, the rates ofdephosphorylation of different RNA substrates seldom vary by more than afactor of 2 (Weber, (1985) Supra). P1 nuclease and mung bean nucleasehave limited preference for certain nucleoside 3'-monophosphates(Shishido & Ando, (1982) Supra). The L-19 IVS RNA, on the other hand,transfers the 3'-phosphate of RNA to a donor molecule with highsubstrate specificity; C₅ p and C₆ p are substrates, whereas pCp and A₆pCp are not. This length and sequence specificity is explained by therequirement that the substance must bind to the enzyme by Watson-Crickbase pairing to the guanosine-rich active site (FIG. 15). If this modelis correct, it should be possible to alter the sequence specificity ofthe phosphotransferase by site-specific mutagenesis of the active site.

A series of sequence-specific 3'-phosphate dephosphorylating enzymeswould provide a useful tool for RNA biochemistry and recombinant RNAmanipulations. However, the RNA enzyme described here hascleavage-ligation activity as well as the dephosphorylation activity.Unless these activities can be separated, the usefulness of the enzymeas a reagent for dephosphorylation of RNA is limited.

Endoribonuclease or "RNA Restriction Endonuclease" Activity

We describe here a fifth enzymatic activity of the Tetrahymena ribozyme.It cleaves other RNA molecules at sequences that resemble the 5' splicesite of the rRNA precursor. Cleavage is concomitant with addition of afree guanosine nucleotide to the 5' end of the downstream RNA fragment;thus, one product can be readily end-labeled during the reaction Thereaction is analogous to the first step of pre-rRNA self-splicing (FIG.1). Cleavage does not require nucleotide G⁴¹⁴ of the ribozyme; thus,unlike the first four activities, it does not involve formation of acovalent enzyme-substrate intermediate. Thus there exists as a result ofthe work of the invention sequence-specific endoribonucleases,protein-free i.e. able to act in the absence of protein, and composed ofRNA, which are enzymatically active on other RNA molecules. These RNAribozymes act on exogenous RNA. Thus the enzyme or ribozyme is composedof RNA and the substrate is RNA (or mixed RNA-DNA polymers).

The ribozyme has high specificity for cleavage after the nucleotidesequence CUCU; under stringent conditions it can discriminate againstsites that have a 3-out-of-4 match to this recognition sequence. Forexample, in a solution containing 2.5M urea the ribozyme cleaves afterCUCU while ignoring the related sequences CUGU and CGCU (FIG. 3c). Thesequence specificity approaches that of the DNA restrictionendonucleases (Nathans, D. and Smith, H. O. (1975) Annu. Rev. Biochem.44:273-293). We further show that site-specific mutations in the activesite of the IVS RNA, the so-called internal guide sequence (Davies, R.W., et al. (1982) Nature 300:719-724; Waring, R. B , et al. (1986)Nature 321:133-139) or 5' exon-binding site (Inoue, T., et al. (1985)Cell 43:431-437; Garriga, G , et al. (1986) Nature 322:86-89; Been, M.D. and Cech, T. R., (1986) Cell 47, 207-216), alter the sequencespecificity of the ribozyme in a predictable manner. In itsendoribonuclease mode, the L-19 IVS RNA recognizes four or morenucleotides in choosing a reaction site. Protein ribonucleases that areactive on single-stranded RNA substrates have specificity only at themononucleotide level (for example, ribonuclease T₁ cleaves afterguanosine). Thus the L-19 has more base-sequence specificity forsingle-stranded RNA than any known protein ribonuclease, and mayapproach the specificity of some of the DNA restriction endonucleases.An attractive feature of this new RNA enribonuclease is that itssubstrate specificity can be completely and predictably changed byaltering the sequence of the internal binding site.

The endoribonuclease reaction is analogous to the first step of pre-rRNAself-splicing (FIG. 1). Both the enzymatic and the self-splicingreactions make use of the same two binding sites, anoligopyrimidine-binding site and a guanosine-binding site, to achievespecificity and to contribute to catalysis. The oligopyrimidine-bindingsite is also known as the 5' guide sequence (Waring, R. B., et al.(1986) Nature 321:133-139) or the 5' exon-binding site (Inoue, T., etal. (1985) Cell 43:431, Garriga, G., et al. (1986) Nature 322:86-89,Been, M. D. and Cech, T. R., Cell (1986) 47, 207-216). Its role inself-splicing has been conclusively demonstrated by the analysis ofsingle-base mutations and second-site suppressor mutations (Waring, R.B., et al. (1986) Supra; Been, M. D. and Cech, T. R., (1986) Supra;Perea, J. and Jacq, C., (1985) EMBO, J. 4:3281). The role of these samenucleotides in the endoribonuclease reaction is demonstrated by thechange in substrate specificity of the mutant enzymes (FIG. 3), thealtered specificity being predictable by the rules of Watson-Crick basepairing. The guanosine-binding site involved in self-splicing has notbeen localized to a particular set of nucleotides, but its generalfeatures have been described (Bass, B. L. and Cech, T. R., (1984) Nature308:820; (1986) Biochemistry 25:4473). The endoribonuclease activityappears to make use of the same guanosine-binding site by the followingcriteria: in both cases guanosine is as active as GTP, whereas UTP, CTP,ATP and dGTP have little if any activity. In addition, the K_(m) of 44uM for GTP shown is in reasonable agreement to the value of 32±8 uMdetermined for self-splicing under somewhat different reactionconditions (Bass, B. L. and Cech, T. R., Biochemistry (1986) Supra).

The endoribonuclease activity of the L-19 IVS RNA does not require its3'-terminal guanosine (G⁴¹⁴). In this respect it differs from thenucleotidyl transfer, phospho transfer and hydrolytic activities of thesame enzyme. In those reactions G⁴¹⁴ participates in a covalentenzyme-substrate complex that appears to be an obligatory reactionintermediate (Zaug, A. J. and Cech, T. R. (1986) Science 231:470-475;Zaug, A. J. and Cech, T. R. (1986) Biochemistry 25:4478). Thus, the L-19IVS RNA is not restricted to reaction mechanisms involving formation ofa covalent enzyme-substrate intermediate. It an also catalyzebisubstrate reactions by a single-displacement mechanism. RibonucleaseP, an RNA enzyme that catalyzes cleavage by hydrolysis rather than bytransesterification, also appears to act without formation of a covalentintermediate (Marsh, T. L., et al. (1985) Science 229:79-81;Guerrier-Takeda, C., et al. (1986) Biochemistry 25:1509).

The L-19 IVS RNA endoribonuclease activity reported here appears torequire single-stranded RNA substrates. Based on work recently reportedby Szostak ((1986) Nature 322:83-86), it seems possible that a smallerversion of the Tetrahymena IVS RNA missing its 5' exon-binding site mayhave an endoribonuclease activity that requires a base-paired substrate.The substrate tested by Szostak ((1986) Nature Supra) was an RNAfragment containing the end of the 5' exon paired with the 5'exon-binding site. However, this RNA "substrate" also included asubstantial portion of the IVS RNA, so it remains to be establishedwhether the ribozyme has endoribonuclease activity with double-strandedRNA substrates in general.

Potential usefulness of sequence-specific RNA endoribonucleases.Sequence-specific endoribonucleases might have many of the sameapplications for the study of RNA that DNA restriction endonucleaseshave for the study of DNA (Nathans, D. and Smith, H. O., (1975) Ann.Rev. Biochem. 44:273). For example, the pattern of restriction fragmentscould be used to establish sequence relationships between two relatedRNAs, and large RNAs could be specifically cleaved to fragments of asize more useful for study. The 4-nucleotide specificity of the ribozymeis ideal for cleavage of RNAs of unknown sequence; an RNA of randomsequence would have an average of 1 cleavage site every 256 bases. Inaddition, the automatic end-labelling of one fragment during ribozymecleavage is a practical advantage.

Development of the ribozymes as useful tools for molecular biology hasbegun. The efficiency of cleavage of large RNA substrates needs to beincreased so that complete digests rather than partial digests can beobtained. The effects of denaturants such as urea and formamide must befurther explored; they appear to increase the sequence specificity ofcleavage, and at the same time they should melt structure in thesubstrate to maximize the availability of target sequences. Finally,mutagenesis of the active site of the ribozyme by those skilled in theart can be accomplished to ascertain all possible permutations of the256 possible tetranucleotide cleavage enzymes.

RNA sequence recognition. Protein ribonucleases can cleave RNAsubstrates with high specificity by recognizing a combination of RNAstructure and sequence, with the emphasis on structure (e.g., RNase III(Robertson, H. D. (1982) Cell 30:669) and RNase M5 (Stahl, D. A., et al.(1980) Proc. Natl. Acad. Sci. USA 77:5644). Known proteins that cleavesingle-stranded RNA substrates, on the other hand, have specificity onlyat the mononucleotide or dinucleotide level (e.g., RNase T₁ cleavesafter guanosines [Egami, F., et al. (1980) Molec Biol. Biochem. Biophys.32:250-277]. Thus, the L-19 IVS RNA has considerably more base-sequencespecificity for cleaving single-stranded RNA than any known proteinribonuclease.

Variant Ribozymes (or other versions of the ribozyme that retainactivity)

Earlier work on sequence requirements for self-splicing (Price, J. V.,et al. (1985) Nucleic Acid. Res. 13:1871) show that sequencerequirements can be examined as shown therein by insertions anddeletions to obtain other self-splicing IVS RNA's. In like manner, wecould alter the L-19 IVS RNA to obtain an array of RNA sequence-specificendoribonuclease molecules. Thus three regions were found by Price etal. to be necessary for IVS self-splicing. Similar experiments wouldreveal necessary portions of L-19 IVS RNA for endoribonuclease activity.Burke, J. M., et al. (1986) Cell 45:167-176 show the role of conservedelements for the IVS self-splicing sequence; that work shows a furtheruse of mutagenesis experiments to alter the highly conserved sequencesto alter activity thereof. Just so, in like manner, the activity of theL-19 IVS RNA can be altered with accompanying alteration in activity toeffect an array of endoribonucleases.

Cech, T. R., et al. have recently found a yet smaller piece of the L-19IVS RNA which contains full enzymatic activity and comprises nucleotides19-331 of the RNA. It is also found that the 21-331 piece is fullyactive. Plasmids have been constructed to produce the L-19 and L-21 IVSRNA strands directly. Here the promoter is moved to the 19 position or21 position and the DNA coding for the restriction site is at the 331position instead of the 414 site.

Been, M. D and Cech, T. R. ((1986) Cell 47:207) show alteration in thespecificity of the polymerase activity to effect polymerase activitywith respect to oligo U using site-specific mutagenesis. Thus thoseskilled in the art can readily use the above to obtain other active L-19IVS RNA enzymes.

Waring, R. B. and Davies, (1984) Gene 28:277 show a class of IVS RNAmolecules with similar structure. This work is similar to that of Cech,T. R., et al. (1983) Proc. Natl. Acad. Sci. USA 80:3903 showing a classof fungal mitochondrial RNA IVS molecules. Some of these other IVSmolecules have been found to be self-splicing. (Cech, T. R., et al.(1981) Cell 27:487; Kruger, K., et al. (1982) ibid. 31:147; Garriga, G.,et al. (1984) ibid 39:631; Van der Horst, G., et al. (1985) ibid 40:759;Chu, F. K., et al. (1985) J. Biol. Chem. 260:10680; Peebles, C. L., etal. Cell in press; Van der Veen, R., et al., ibid. in press) Thus aseries, or many series or class, or family of endoribonucleases from thesame or other natural sources can be based on the work of the invention.Those skilled in the art will be able to search out other RNA enzymesfrom various natural sources.

The following RNA sequence elements can be considered to provide theminimum active site for ribozyme activity, based on (Cech, et al., PNAS(1983) and Waring & Davies Supra). Elements A and B interact with eachother, as do 9L and 2. In many positions of the sequence, more than 1base is allowed; the observed substitutions are shown by printing aletter directly below the base for which it can substitute. For example,at position 1 in sequence element A, the nucleotides A and U areobserved, A being more common. ##STR2##

The linear sequence of non-template DNA coding for L IVS RNA is shownbelow. Coding for the L-19 IVS RNA "ribozyme" begins at the siteindicated by the arrow and extends to the end (G⁴¹⁴). Of course, in RNAthe T's are U's. ##STR3##

In the discussion of L-19 IVS RNA, the region of the active sitesequence discussed with respect to activity and variants is: ##STR4##

The first ribonucleotide for the L-19 IVS RNA at the 5'OH end is theequivalent of the nucleotide 20 of the intact L IVS RNA. As regardspositions 23, 24, 25 etc., there are positions 23, 24, 25 of the L IVSRNA (as if the first 19 positions were present).

Since dC₅ binds to the L-19 IVS RNA (see above), it is likely that theendoribonucleases will work on mixed polymers of RNA and DNA. Forexample, L-19 IVS RNA will bind the DNA portion while the RNA enzymeworks on the RNA piece of the mixed polymer. Alteration of the bindingsite to bind the other nucleotides will result in an array of mixedpolymer activity in a series of such endoribonucleases.

Abbreviations: IVS, intervening sequence; L-19 IVS RNA (read "L minus19"), a 395-nucleotide linear RNA missing the first 19 nucleotides ofthe IVS; CHES,2-(cyclohexylamino)ethanesulfonic acid; EDTA,ethylenediaminetetraacetic acid; MES, 2-(N-Morpholino)-ethanesulfonicacid; Tris, tris(hydroxymethyl)aminomethane; p^(*), ³² P within anoligonucleotide (for example, C₅ p^(*) is CpCpCpCpC[³² P]-pCp).

Enzyme Preparation. L-19 IVS RNA can be synthesized and purified asdescribed by Zaug and Cech (1986) Science (Wash., D.C.) 231:470-475 (seeFIG. 5 for detailed description). In brief, RNA was transcribed frompSPTT1A3 with bacteriophage SP6 RNA polymerase in vitro. (AlternativelyRNA can be transcribed from pT7-TT1A3 or from any of the plasmids in thepBG series with bacteriophage T7 RNA polymerase in vitro). Transcriptswere further incubated to promote self-splicing and cyclization of theIVS RNA. The RNA was subsequently incubated in MgCl₂ at pH 9.0(site-specific hydrolysis conditions) to convert circular IVS RNA toL-19 IVS RNA. The L-19 IVS RNA was purified by polyacrylamide gelelectrophoresis and Sephadex G-50 chromatography. Enzyme concentrationwas determined by spectrophotometry assuming a molar extinctioncoefficient at 260 nm of 3.26×10⁶ M⁻¹ cm⁻¹.

Active plasmids in use are as follows:

pSPTT1A3, pT7-TT1A3, pBGST7, pBG/-2G:23C, pBG/23C, pBG/-3G:24C, pBG/24C,pBG/-4G:25C, pBG/25C, and pBG/23A₄ and pT7L-21. The PBG plasmid seriesis described in Been, M. and Cech, T. R. (1986) Cell 407:207. Forexample, the pBG/3G:24C and the pBG/24C₅ plasmids produce the L-19 IVSRNA 24C variant which cleaves RNA after the CGCU 4 base sequence. Theseplasmids are on deposit and available at the Department of Chemistry andBiochemistry, University of Colorado, Boulder, Col. 80309-0215. Examplesof these including pBG ST7 (ATCC 40288), pT7-TT1A3 (ATCC 40290) andpBG/-3G:24C (ATCC 40289) have been deposited with the American TypeCulture Collection (ATCC) 12301 Parklawn Drive, Rockville Md. 20301 onNov. 25, 1986. The plasmid pT7L-21 makes the L-21 IVS RNA wherein thefirst 21 bases are deleted. This plasmid (ATCC 40291) was also placed ondeposit at the ATCC on Dec. 2, 1986.

Preparation of Substrates. C₅ p^(*) Cp and A₆ p^(*) Cp were preparedfrom C₅ --OH and A₆ --OH, respectively, with T₄ RNA ligase (New EnglandNuclear), p^(*) Cp, and ATP. Products were purified by 20%polyacrylamide-7M urea gel electrophoresis and Sephadex G-25chromatography. C₅ p^(*) was prepared from C₅ p^(*) Cp by treatment withcalf intestinal phosphatase and beta-elimination (Winter & Brownlee,1978 Nucleic Acid. Res. 3129). Unlabeled C₅ p was prepared in a similarmanner with unlabeled pCp as donor in the ligase reaction. Concentrationwas determined by spectrophotometry using a molar extinction coefficientat 270 nm of 30×10³ M⁻¹ cm⁻¹.

Preparation of E-p^(*). Unlabeled L-19 IVS RNA (16 pmol) was incubatedwith 5.2 pmol of C₅ p^(*) in 50 mM NaOAc, pH 5.0, and 20 mM MgCl₂ at 42°C. for 10 min. The reaction was stopped by the addition of EDTA to 40mM. The E-p^(*) was purified from unreacted C₅ p^(*) by columnchromatography on Sephadex G-100-120, which was equilibrated in 0.01MTris-HCl, pH 7.5, 0.25M NaCl, and 0.001M EDTA. The fractions thatcontained E-p^(*) complex were pooled and precipitated with 3 volumes ofethanol. The dried precipitate was then dissolved in H₂ O.

Standard Nucleotidyl Transferase Reaction Conditions

The oligoribonucleotide substrate (for example 10-100 uM pC₅) isincubated with ribozyme (for example 0.1-2.0 uM L-19 IVS RNA) at 42° C.in 20 mM Mgcl₂ and 50 mM Tris, pH 7.5 for 1 hour.

Standard Transphosphorylase Conditions

The 3'-phosphorylated RNA substrate (for example 2.5 uM C₅ p) isincubated with ribozyme (for example 0.1 uM L-19 IVS RNA) and anacceptor oligonucleotide (for example, 200 uM UpCpU) at 42° C. in 20 mMMgCl₂ and 50 mM MES, pH 6.0 for 3 hours.

Standard Acid Phosphotase Conditions

The 3' phosphorylated RNA substrate (for example 2.5 uM C₅ p) incubatedin equimolar concentration of ribozyme (for example 2.5 uM L-19 IVS RNA)at 42° C. in 20 mM MgCl₂ and 50 mm NaC₂ H₃ O₂ (Na acetate), pH 5.0 for24 hours.

Standard Endoribonuclease Reactions

Substrate RNA is pretreated with glyoxal according to the procedure ofCarmichael and McMaster (1980) Meth in Enzymol. 65:380-391 and thenethanol precipitated and the precipitate pelleted by centrifugation inan eppendorf centrifuge. The pellet is dried and the RNA re-suspended inwater. The glyoxylated substrate RNA (0.2 uM) is incubated with ribozyme(for example L-19 IVS-beta RNA, 0.2 uM) at 50° C. in 10 mM MgCl , 10 mMNaCl, 50 mM Tris-HCl pH 7.5, 0.5 mM GTP, 2.5M Urea for 1 hour.

Stopping Reactions and Analyzing Products

In all cases reactions are stopped by the addition of EDTA to a finalconcentration of 25 mM. Products can be analyzed by electrophoresis in a20% polyacrylamide, 7.0M Urea gel (standard sequencing gel). If ³²P-labelled RNA substrates are used, products can be localized byautoradiography.

The following Examples and the standard conditions above serve toillustrate, but not to limit the invention.

EXAMPLE I

Sequence-specific cleavage of large RNAs:

The L-19 IVS RNA enzyme was prepared by incubation of pre-rRNA underconditions that promote self-splicing, cyclization, and site-specifichydrolysis, (Zaug, A. J., et al. (1984) Science 224:574; Zaug, A. J., etal. (1986) Science 231:470). The 3'-terminal guanosine (G⁴¹⁴) was thenremoved from the L-19 IVS RNA by periodate oxidation followed bybeta-elimination (Winter, G., et al. (1978) Nucleic Acids Res.5:3129-3139). As expected, the resulting ribozyme (L-19 IVS-beta) hasgreatly reduced activity as a nucleotidyl-transferase, assayed using [³²P]-p(C)₅ as a substrate. When GTP was added, however, the ribozyme wasable to cleave p(C)₅ as well as large RNA molecules. For example, the504 nt pAK105 transcript (Mount, S. M., et al. (1983) Cell 33:509-518),a fragment of mouse beta-globin pre-mRNA containing the first intron,was cleaved to give major fragments of 148, 360 and 464 nt, as well assome minor fragments (FIG. 2a). As shown below, the 360 and 148 ntfragments can be explained as the 5' and 3' products of cleavage atposition 360. The 464 nt fragment is the 3' product of cleavage atposition 44, the 5' 44 nt fragment being to small to be observed. Theabsence of a major amount of a 316 nt RNA, the expected product ofcleavage at both position 44 and 360, is indicative of partial digestionwith few molecules cleaved more than once.

Cleavage required magnesium ion (optimum at 10-20 mM MgCl₂) and wasessentially independent of monovalent cation the range 0-200 mM NaCl.The pH optimum was in the range of 7.5-8.0, and the temperature optimumwas approximately 50° C. Although the beta-eliminated L-19 IVS RNA wascompetent to catalyze the cleavage reaction, removal of G⁴¹⁴ from theribozyme was not required for cleavage activity. The enzyme worked atthe same rate whether or not G⁴¹⁴ had been removed. We explain theactivity of the intact L-19 IVS RNA by the postulate that, at saturatingconcentrations of GTP, the attack by GTP on the substrate competes veryeffectively with attack by G⁴¹⁴.

We note the IVS RNA and L-19 IVS RNA are protein-free. The L-19 IVS RNAis therefore a protein-free RNA enzyme (or ribozyme). L-19 IVS RNA andthe like also function as enzymes in the absence of proteins. Thisapplies to exogenous as well as endogenous protein. This is evidenced byretention of activity when subjected to protease activity, boiling orsodium dodecyl sulfate. Any RNA polymerase protein from thetranscription system used to produce IVS RNA is removed by phenolextraction. The ability to make the IVS RNA in a totally defined systemin vitro and remove the RNA polymerase by phenol extraction is furtherevidence of the protein-free nature of the reaction.

EXAMPLE II

Labelled cleavage products:

When [alpha-³² P]GTP was included in the reaction of pAK105 RNA as inExample I above, the 148 and 464 nt cleavage products were labeled (FIG.2b). Direct sequencing of these labeled RNA fragments (e.g., FIG. 2c)showed that cleavage and GTP-addition occur at nucleotides 44 and 360 inthe sequence, such that the downstream cleavage products are 5'-GTPlabeled. The bonds formed by GTP addition are sensitive to RNase T₁,confirming that the GTP was covalently added through its 3'-0 by anormal 3'-5' phosphodiester bond. Reaction of the 841 nt pT7-1 (Xmn 1)RNA (essentially pBR322 sequences) produced 4 major labeled fragments.These were sequenced in the same manner. pT7-1 RNA with an additional122 nt at its 3' end, produced by transcription of pT7-1 DNA that hadbeen cleaved at the Sca I site, showed the expected increase inmolecular weight of the labeled products. In all of the reactions,including those in which substrate RNA was omitted, the L-19 IVS RNAbecame labeled, perhaps by the guanosine-exchange reaction proposedelsewhere (Zaug, A. J., et al. (1985) Science 229:1060-1064; Price, J.V., et al. (1987) J. Mol. Biol. in press). The sites of self-labelingwere heterogeneous.

EXAMPLE III

Specificity Assessment:

The sequence near the 5' end of each end-labeled product (See Example IIabove) was compared to the known sequence of the RNA to identify thenucleotides preceding the site of cleavage. The results are summarizedin Table 1. Both the major and the minor cleavage sites are preceded byfour pyrimidines, the consensus sequence being CUCU. This is exactly thetetranucleotide sequence expected to be an optimal target for theribozyme. The importance of nucleotides at positions -5 and -6 relativeto the cleavage site is not yet clear, although the absence of Gresidues may be significant. There is no apparent sequence preferencedownstream from the cleavage site, with all four nucleotides representedat position +1.

In assessing specificity, it is also necessary to consider whichpotential sites in the RNA were not cleaved by the ribozyme. For thepAK105 RNA, there was only one CUCU site at which cleavage was notobserved. (Cleavage at this site would have produced a labeled 378 ntRNA.) On the other hand, a great many sites that match CUCU in 3 out of4 positions were not cleaved. These include 17 CUMU sequences (whereM≠C) and 7 CNCU sequences (where N≠U). In pT7-1 RNA, cleavage wasobserved at both the CUCU sequences in the RNA, but at only one of the15 UUUU sequences present. Thus, the ribozyme has a strong preferencefor cleavage after the tetranucleotide CUCU.

EXAMPLE IV

Cleavage within regions of base-paired RNA secondary structure:

M1 RNA, the RNA subunit of E. coli RNase P (Reed, R. E., et al. (1982)Cell 30:627-636), was not cleaved by L-19 IVS RNA-beta under standardreaction conditions (FIG. 2b). M1 RNA contains the sequence UCCUCU,which should be an excellent targent site. However, this sequence isinvolved in a stable hairpin stem (Guerrier-Takada, C., et al. (1984)Biochemistry 23:6327-6334; Pace, N. R., et al. (1985) Orig. of Life16:97-116), which presumably makes it unavailable as a substrate. Wehave found that denaturation of M1 RNA with glyoxal allowed efficientcleavage of this site by L-19 IVS RNA, in support of the interpretationthat RNA secondary structure can inhibit cleavage. The glyoxal procedureused for denaturation is according to Carmichael, G. G., et al. (1980)Meth. in Enzymol 65:380-391.

EXAMPLE V

Active-site mutations alter substrate specificity:

The substrate specificity was next studied in a defined system where wecould be certain that secondary structure in the substrate RNA was notaffecting the cleavage reaction. Oligoribonucleotide substrates weresynthesized by the phage T7 RNA polymerase transcription methoddeveloped by Uhlenbeck and co-workers (Lowary, P., et al. (1986) NATOASI Series, vol. 110, 69-76) (FIG. 3a). One substrate contained aperfect match to the tetranucleotide consensus sequence. Two othersubstrates had single-base changes giving a 3-out-of-4 match to theconsensus.

These substrates were tested with the wild-type L-19 IVS RNA and withtwo altered ribozymes (Been, M. D. and Cech, T. R., (1986) Cell47,207-216). The two variants have single-base changes in the 5'exon-binding site that alter the sequence specificity of the first stepin pre-rRNA self-splicing (Been, M. D., et al. Supra) The 23C variant (Gconverted to C at position 23 of the L-19 IVS RNA) is expected torecognize CUGU substrates, and the 24C (A converted to C at position 24)variant should recognize CGCU (FIG. 3b). In the course of these studieswe found that the inclusion of 2.5M urea or 15% formamide in thereactions greatly increased the specificity, allowing each ribozyme todifferentiate substrates with a single base change in the recognitionsequence. Our operating model is that these denaturants destabilized thebase-pairing between the substrate and the active site nucleotides ofthe ribozyme, thereby discriminating against mismatched complexes. Theresults of treatment of the 3 substrates with each of the 3 ribozymes inthe presence of 2.5M urea are shown in FIG. 3c. Each substrate iscleaved exclusively by the ribozyme that is capable of making aperfectly base-paired enzyme-substrate complex (FIG. 3b). Thus it iscontemplated the active site can be manipulated to recognize any basesequence so far that is XYZU, where X, Y and Z can be any of the fourbases A,U,C,G and the nucleotides in the four base sequence can be thesame or different.

When variant ribozymes were incubated with the 504 nt pAK105 RNA, eachribozyme gave a different pattern of cleavage products. One majorcleavage site has been mapped for three variant ribozymes, including a25C variant (G converted to C at position 25). The sites cleaved by the23C, 24C and 25C ribozymes are preceded by CCCUGU, UCUGCU, and CUGUCU,respectively; the underlining indicates the base that would form a G.Cbase pair with the mutated nucleotide in the variant ribozyme. Each ofthese sequences can form 6 continuous base-pairs with the active sitenucleotides of the variant ribozyme. While more cleavage sites must besequenced before specificity can be properly assessed, these initialresults are promising.

EXAMPLE VI

Cleavage is catalytic:

The time course of cleavage of the oligonucleotide substrateGGCCCUCU^(*) AAAAA (where the asterisk designates the cleavage site) bythe wild-type ribozyme is shown in FIG. 4a. The reaction of 2.5 uMsubstrate with 0.2 uM ribozyme is 66% complete in 90 minutes. Thus, itis readily apparent that the ribozyme is acting catalytically.

The reaction rate was determined at a series of substrateconcentrations. The kinetics are adequately described by theMichaelis-Menten rate law. The dependence of the rate on GTPconcentration is shown in the form of a Lineweaver-Burk plot in FIG. 4b.The K_(m) for GTP is 44 uM. The dependence of the rate on RNA substanceconcentration at saturating GTP concentration is shown in FIG. 4C. TheK_(m) for this oligoribonucleotide substrate is 0.8 uM, and k_(cat) is0.13 min⁻¹. Thus under Vmax conditions the enzyme turns over about 8times per hour.

EXAMPLE VII

The L-19 IVS RNA catalyzes the cleavage and rejoining ofoligonucleotides:

Unlabeled L-19 IVS RNA was incubated with 5'-³² P-labeled pC₅ in asolution containing 20 mM MgCl₂, 50 mM tris-HCl, pH 7.5. The pC₅ wasprogressively converted to oligocytidylic acid with both longer andshorter chain length than the starting material (FIG. 5A). The longerproducts extended to at least pC₃₀, as judged by a longer exposure of anautoradiogram such as that shown in FIG. 5A. THe shorter products wereexclusively pC₄ and pC₃. Incubation of pC₅ in the absence of the L-19IVS RNA gave no reaction (FIG. 5C).

Phosphatase treatment of a 60-minute reaction mixture resulted in thecomplete conversion of the ³² P radioactivity to inorganic phosphate, asjudged by polyethyleneimine thin-layer chromatography (TLC) in 1M sodiumformate, pH 3.5 (Zaug, A., et al. unpublished data). Thus, the5'-terminal phosphate of the substrate does not become internalizedduring the reaction, and the substrate is being extended on its 3' endto form the larger oligonucleotides. When C₅ p^(*) C was used as thesubstrate and the products were treated with ribonuclease T₂ orribonuclease A, the ³² P radioactivity was totally converted to Cp^(*)(Zaug, A., et al. unpublished data). Thus, the linkages being formed inthe reaction were exclusively 3',5'-phosphodiester bonds. The productsof the C₅ p^(*) C reaction were totally resistant to phosphatasetreatment.

The reaction was specific for ribonucleotides, no reaction taking placewith d-pC₅ (FIG. 1B) or d-pA₅ (Zaug, A., et al. unpublished data). Amongthe oligoribonucleotides, pU₆ was a much poorer substrate than pC₅ orpC₆ (FIG. 5D), and pA₆ and gave no reaction (Zaug, A., and Cech, T. R.,(1986), Biochemistry 25:4478).

No reaction occurred when magnesium chloride was omitted. The enzymeactivity was approximately constant in the range 5 to 40 mM MgCl₂ (Zaug,A., et al. unpublished data). The 20 mM concentration was routinely usedto circumvent the potential effect of chelation of Mg²⁺ by highconcentrations of oligonucleotide substrates.

The L-19 IVS RNA is regenerated after each reaction, such that eachenzyme molecule can react with many substrate molecules. For example,quantitation of the data shown in FIG. 5G revealed that 16 pmol ofenzyme converted 1080 pmol of pC₅ to products in 60 minutes. Suchnumbers underestimate the turnover number of the enzyme; because theinitial products are predominantly C₆ and C₄, it is likely that theproduction of chains of length greater than six or less than fourinvolves two or more catalytic cycles. Quantitation of the amount ofradioactivity in each product also provides some indication of thereaction mechanism. At early reaction times, the amount of radioactivity(a measure of numbers of chains) in products larger than pC₅ isapproximately equal to that found in pC₄ plus pC₃, consistent with amechanism in which the total number of phosphodiester bonds is conservedin each reaction. As the reaction proceeds, however, the radioactivitydistribution shifts toward the smaller products. This is most likely dueto a competing hydrolysis reaction also catalyzed by the L-19 IVS RNA,as described below.

The rate of conversion of 30 uM pC₅ to products increases linearly withL-19 IVS RNA enzyme concentration in the range 0.06 to 1.00 uM (Zaug,A., et al. unpublished data). At a fixed enzyme concentration (FIG. 5, Eto G), there is a hyperbolic relation between the reaction rate and theconcentration of pC₅. The data are fit by the Michaelis-Menten rate lawin FIG. 6. The resulting kinetic parameters are K_(m) =42 uM and k_(cat)=1.7 min⁻¹.

The stability of the enzyme was determined by preliminary incubation at42° C. for 1 hour in the presence of Mg²⁺ (standard reaction conditions)or for 18 hours under the same conditions but without Mg²⁺. In bothcases, the incubated enzyme had activity indistinguishable from that ofuntreated enzyme tested in parallel, and no degradation of the enzymewas observed on polyacrylamide gel electrophoresis (Zaug, A., et al.unpublished data). Thus the L-19 IVS RNA is not a good substrate. Theenzyme is also stable during storage at -20° C. for periods of months.The specific activity of the enzyme is consistent between preparations.

EXAMPLE VII

Convalent intermediate. When C₅ p^(*) was used as a substrate,radioactivity became covalently attached to the L-19 IVS RNA (FIG. 7A)(The radioactive phosphate was bonded covalently to the L-19 IVS RNA asjudged by the following criteria: it remained associated when thecomplex was isolated and subjected to a second round of denaturing gelelectrophoresis; it was released in the form of a mononucleotide uponRNase T₂ treatment; and it was released in the form of a series ofunidentified oligonucleotides upon RNase T₁ treatment (A. Zaug and T.Cech, unpublished data). These results are consistent with a series ofcovalent enzyme-substrate complexes in which various portions of C₅p^(*) C are linked to the L-19 IVS RNA via a normal 3'-5'-phosphodiesterbond. This observation, combined with our previous knowledge of themechanism of IVS RNA cyclization (Zaug, A. J., et al., (1984) Science224:574; Sullivan and Cech, T. R., (1985) Cell 42:639; Zaug, A. J., etal. (1983) Nature (London) 301:578), Been, M. and Cech T. R. (1985)Nucleic Acids Res. 13:8389), led to a model for the reaction mechanisminvolving a covalent enzyme-substrate intermediate (FIG. 8).

This reaction pathway is supported by analysis of reactions in which atrace amount of p^(*) C₅ was incubated with a large molar excess of L-19IVS RNA. The cleavage reaction occurred with high efficiency, as judgedby the production of p^(*) C₄ and p^(*) C₃, but there was very littlesynthesis of products larger than the starting material (FIG. 7B;compare to FIG. 5A). These data are easily interpreted in terms of theproposed reaction pathway. The first step, formation of the covalentintermediate with release of the 5'-terminal fragment of theoligonucleotide, is occurring normally. The first step consumes all thesubstrate, leaving insufficient C₅ to drive the secondtransesterification reaction.

The model shown in FIG. 8 was tested by isolating the covalentenzyme-substrate complex prepared by reaction with C₅ p^(*) C andincubating it with unlabeled C₅. A portion of the radioactivity wasconverted to oligonucleotides with the electrophoretic mobility of C₆,C₇, C₈, and higher oligomers (FIG. 7C). In a confirmatory experiment,the covalent complex was prepared with unlabeled C₅ and reacted withp^(*) C₅. Radioactivity was again converted to a series of highermolecular weight oligonucleotides (Zaug, A., et al. unpublished data).In both types of experiments the data are readily explained if thecovalent complex is a mixture of L-19 IVS RNA's terminating in . . .GpC, . . . GpCpC, . . . GpCpCpC, and so on. Because they can react withC₅ to complete the catalytic cycle, these covalent enzyme-substratecomplexes are presumptive intermediates in the reaction (FIG. 8). A moredetailed analysis of the rate of their formation and resolution isneeded to evaluate whether or not they are kinetically competent to beintermediates. We can make no firm conclusion about that portion of theenzyme-substrate complex that did not react with C₅. This unreactive RNAcould be a covalent intermediate that was denatured during isolationsuch that it lost reactivity, or it could represent a small amount of adifferent enzyme-substrate complex that was nonproductive and thereforeaccumulated during the reaction.

The G⁴¹⁴ -A¹⁶ linkage in the C IVS RNA, the G⁴¹⁴ -U²⁰ linkage in the C'IVS RNA, and the G⁴¹⁴ -U⁴¹⁵ linkage in the pre-rRNA are unusualphosphodiester bonds in that they are extremely labile to alkalinehydrolysis, leaving 5' phosphate and 3'-hydroxyl termini (Zaug, A. J. etal, (1984) Science 224:574 and Inoue, T., et al. (1986) J. Mol. Biol.189,143-165). We therefore tested the lability of the G⁴¹⁴ -C linkage inthe covalent enzyme-substrate intermediate by incubation at pH 9.0 in aMg²⁺ -containing buffer. This treatment resulted in the release ofproducts that comigrated with pC and pCpC markers and larger productsthat were presumably higher oligomers of pC (FIG. 7D). Thin-layerchromatography was used to confirm the identity of the major products(Zaug, A., et al. unpublished data). In those molecules that releasedpC, the release was essentially complete in 5 minutes. Approximatelyhalf of the covalent complex was resistant to the pH 9.0 treatment. Onceagain, we can make no firm conclusion about the molecules that did notreact. The lability of the G⁴¹⁴ -C bond forms the basis for the L-19 IVSRNA acting as a ribonuclease (FIG. 8) by a mechanism that is distinctfrom that of the endoribonuclease described in Examples I through VI.

A competitive inhibitor. Deoxy C₅, which is not a substrate for L-19 IVSRNA-catalyzed cleavage, inhibits the cleavage of pC₅ (FIG. 9A). Analysisof the rate of the conversion of pC₅ to pC₄ and pC₃ as a function ofd-C₅ concentration is summarized in FIG. 9B and C. The data indicatethat d-C₅ is a true competitive inhibitor with the inhibition constantK_(i) =260 uM. At 500 uM, d-A₅ inhibits the reaction only 16 percent asmuch as d-C₅. Thus, inhibition by d-C₅ is not some general effect ofintroducing a deoxyoligonucleotide into the system but depends onsequence.

The formation of the covalent enzyme-substrate intermediate (EpC) can berepresented as ##STR5##

If k-₁ >>k₂, then K_(m) =k-₁ /k₁, the dissociation constant for thenoncovalent E·C₅ complex. The observation the the K_(i) for d-C₅ iswithin an order of magnitude of the K_(m) for C₅ can then be interpretedin terms of d-C₅ and C₅ having similar binding constants for interactionwith the active site on the enzyme. This fits well with the idea thatthe substrate binds to an oligopurine (R₅) sequence in the active siteprimarily by Watson-Crick base-pairing, in which case the C₅ ·R₅ duplexand the d-C₅ ·R₅ duplex should have similar stability.

Enzyme mechanism and its relation to self-splicing. The stoichiometry ofthe reaction products (equimolar production of oligonucleotides smallerthan and larger than the starting material), the lack of an ATP or GTP(adenosine triphosphate; guanosine triphosphate) energy requirement, theinvolvement of a covalent intermediate, the specificity for oligoCsubstrates, and the competitive inhibition by d-C₅ lead to a model forthe enzyme mechanism (FIG. 8). The L-19 IVS RNA is proposed to bind thesubstrate noncovalently by hydrogen-bonded base-pairing interactions. Atransesterification reaction between the 3'-terminal guanosine residueof the enzyme and a phosphate ester of the substrate then produces acovalent enzyme-substrate intermediate.

Transesterification is expected to be highly reversible. If the productC₄ rebinds to the enzyme, it can attack the covalent intermediate andreform the starting material, C₅. Early in the reaction, however, theconcentration of C₅ is much greater than the concentration of C₄ ; if C₅binds and attacks the covalent intermediate, C₆ is produced (FIG. 8).The net reaction is 2 C₅ converted to C₆ +C₄. The products aresubstrates for further reaction, for example, C₆ +C₅ is converted to C₇+C₄ and C₄ +C₅ is converted to C₃ +C₆. The absence of products smallerthan C₃ is explicable in terms of the loss of binding interactions of C₃relative to C₄ (C₃ could form only two base pairs in the binding modethat would be productive for cleavage).

The transesterification reactions are conservative with respect to thenumber of phosphodiester bonds in the system. Thus, RNA ligation canoccur without an external energy source as is required by RNA or DNAligase. Hydrolysis of the covalent intermediate competes withtransesterification. The net reaction is C₅ +H₂ O converted to C₄ +pC,with the L-19 IVS RNA acting as a ribonuclease.

On the basis of our current understanding of the reaction, the catalyticstrategies of the L-19 IVS RNA enzyme appear to be the same as thoseused by protein enzymes (Jencks, W. P., (1969) Catalysis in Chemistryand Enzymology (McGraw-Hill, New York). First, the RNA enzyme, likeprotein enzymes, forms a specific noncovalent complex with itsoligonucleotide substrate. This interaction is proposed to hold theoligonucleotide substrate at a distance and in an orientation such as tofacilitate attack by the 3'-hydroxyl of the terminal guanosine of theenzyme. Second, a covalent enzyme-substrate complex is a presumptiveintermediate in the L-19 IVS RNA reaction. Covalent intermediates areprevalent in enzyme-catalyzed group transfer reactions. Third, thephosphodiester bond formed in the covalent intermediate is unusuallysusceptible to hydrolysis, suggesting that it may be strained oractivated to facilitate formation of the pentavalent transition stateupon nucleophilic attack (Zaug, A. J., et al. (1985) Biochemistry24:6211; Zaug, A. J., et al. (1984) Science 224:574). Similarly, proteincatalysts are thought to facilitate the formation of the transitionstate, for example, by providing active site groups that bind thetransition state better than the unreacted substrate (Fersht, A., (1985)Enzyme Structure and Mechanism (Freeman, New York, ed. 2); Wells,T.N.C., et al. (1985) Nature (London) 316:656). Thus far there is noevidence that another major category of enzyme catalysis, generalacid-base catalysis, occurs in the L-19 IVS RNA reactions, but we thinkit likely that it will be involved in facilitating the required protontransfers.

Each L-19 IVS RNA-catalyzed transesterification and hydrolysis reactionis analogous to one of the steps in Tetrahymena pre-rRNA self-splicingor one of the related self-reactions (FIG. 10). Thus, the finding ofenzymatic activity in a portion of the IVS RNA validates the view thatthe pre-rRNA carries its own splicing enzyme as an intrinsic part of itspolynucleotide chain. It seems likely that the C₅ substrate binding siteof the L-19 IVS RNA is the oligopyrimidine binding site that directs thechoice of the 5' splice site and the various IVS RNA cyclization sites(Sullivan, F. X., et al. (1985) Cell Supra; Been, M., et al. (1985)Nucleic Acid Research Supra; Inoue, T., et al. J. Mol. Biol., Supra;Inoue, T., et al. (1985) Cell 43:431. Although the location of this sitewithin the IVS RNA has not been proved, the best candidate is a portionof the "internal guide sequence" proposed by Davies and co-workers(Davies, R. W., et al. (1982) Nature (London) 300:719; Waring, R. B., etal. (1983) J. Mol. Biol. 167:595). Michel and Dujon (Michel, F., et al.(1983) EMBO J. 2:33) show a similar pairing interaction in their RNAstructure model. The putative binding site, GGAGGG, is located atnucleotides 22 to 27 of the intact Tetrahymena IVS RNA and at positions3 to 8 very near the 5' end of the L-19 IVS RNA. If this is thesubstrate binding site, site-specific mutation of the sequence shouldchange the substrate specificity of the enzyme in a predictable manner.

EXAMPLE IX

The L-19 IVS RNA Dephosphorylates C₆ p. When oligo(cytidylic acid) witha 3'-terminal hydroxyl group is incubated with the L-19 IVS RNA enzymein 20 mM MgCl₂, it is converted to oligo(C) with both larger and smallerchain lengths (FIG. 1A) as described previously (Examples VII and VIIIand Zaug & Cech, (1986) Science (Wash., D.C.) 231:470-475. The reactionis specific for oligo(C); for example, pA₆ --OH is unreactive under thesame conditions (FIG. 11A).

When oligo(cytidylic acid) with a 3'-terminal phosphate is incubatedwith excess L-19 IVS RNA in 20 mM MgCl₂, the substrate is converted to aproduct with reduced electrophoretic mobility (FIG. 11A). This abruptreduction in electrophoretic mobility, equivalent to an increase ofapproximately three nucleotides on a sequencing ladder, is exactly thatobtained by treating the substrate with alkaline phosphatase (not shownin FIG. 11; an example is shown in FIG. 12). Thus, it appeared that theproduct was C₆ --OH. When the substrate is internally labeled (C₅ p^(*)Cp), the labeled phosphate is retained in the product (FIG. 11A). On theother hand, when the substrate is terminally labeled (C₅ p^(*)), theoligonucleotide product is unlabeled and the L-19 IVS RNA becomeslabeled (FIG. 11B). These findings confirmed that the reaction involvesremoval of the 3'-terminal phosphate of the substrate.

Dephosphorylation is specific for the 3'-phosphate of oligo(cytidylicacid). The 5'-phosphate of pC₅ is not reactive (FIG. 11A), and neitherphosphate is removed from pCp (data not shown). (We estimate that 0.1%reaction would have been detected). Neither A₆ Cp (FIG. 11A) nor pA₆ p(not shown) is a substrate. (We estimate that 0.1% reaction would havebeen detected). On the basis of this sample, it appears that there isboth a minimum length requirement and a sequence requirements for asubstrate and that the requirement are similar to those of thenucleotidyl transfer activity of the L-19 IVS RNA (Zaug & Cech, (1986)Science Supra).

EXAMPLE X

Formation and Stability of E-p. We next investigated the fate of thephosphate that is removed from C₅ p in the presence of L-19 IVS RNA. Atneutral pH no inorganic phosphate is formed during the reaction, asjudged by thin-layer chromatography of the reaction products (data notshown). When the reaction is conducted in the presence of C₅ p^(*), itbecomes clear that the phosphate is transferred to the L-19 RNA (FIG.11B). Treatment of the phosphorylated L-19 IVS RNA (hereafter calledE-p) with alkaline phosphatase leads to quantitative release of theradioactivity in the form of inorganic phosphate. Thus, thedephosphorylation of the substrate is accomplished bytransphosphorylation. The structure of E-p has been determined; thephosphate is esterified through the 3'-0 of the 3'-terminal guanosineresidue (G414) of the RNA (Zaug and Cech, unpublished results).

The rate of conversion of G₅ p to C₅ --OH+E-p is pH-dependent with anoptimum around pH 5.0. A sample of the data is shown in FIG. 12. Thephospho transfer reaction is essentially pH-independent in the range pH7.5-9 and proceeds at a rate similar to that of the nucleotidyl transferreaction (FIG. 12A). At pH 5 the phospho transfer reaction isaccelerated more than 20-fold, while the nucleotidyl transfer reactionis unaffected. At pH 4, the enzyme still has substantial phosphotransfer activity while its nucleotidyl transfer activity is greatlydiminished. Since the enzyme is probably starting to denature below pH 5(Zaug, et al., (1985) Biochemistry 24:6211) the phospho transferactivity profile in this range presumably reflects the inactivation ofthe enzyme rather than the pH optimum for the transfer step. Data suchas those shown in FIG. 12B were quantified, and the resulting rateconstants are summarized in FIG. 12C.

The covalent intermediate formed during the reaction of pC₅ --OH withthe L-19 IVS RNA (E-pC) is alkali-labile; at pH 9.0 it undergoeshydrolysis, releasing the nucleotide pC and regenerating the free enzyme(Zaug & Cech, (1986) Science Supra). We therefore investigated thestability of the phosphate monoester in the phosphenzyme E-p. There isno detectable hydrolysis of the phosphate monoester at pH 9.0,conditions in which E-pC treated in parallel gave release of pC, or atany other pH in the range 7.0-9.0.

At acidic pH, on the other hand, the terminal phosphate monoester of E-punderwent slow hydrolysis. When E-p was incubated at pH 5.0, thephosphate was released as P_(i) at a rate of approximately 10%/h. Therate was slower at pH 4.0 and at pH 6.0 than at pH 5.0. Release of P_(i)was also observed during the reaction of C₅ p^(*) with L-19 IVS RNA atpH 5.0 at reaction times of 2 and 4h. Thus, the L-19 IVS RNA has acidphosphatase activity. This hydrolytic reaction is so slow, however, thatwe have not attempted to demonstrate multiple turnover.

EXAMPLE XI

Phosphorylation of the Enzyme Is Reversible. When unlabeled E-p isincubated with a trace amount of C₅ p^(*), very little reaction takesplace (FIG. 13A). In contrast, when the same E-p is incubated with atrace amount of labeled pC₅ --OH, labeled pC₅ p is progressively formed(FIG. 13B). The products normally formed by incubation of pC₅ --OH withexcess L-19 IVS RNA, pC₄ --OH and pC₃ --OH (Zaug & Cech, 1986), are notobserved. Thus, E-p is inactive as a nucleotidyltransferase but isreadily subject to a reverse phosphorylation reaction.

The reversibility of phosphorylation of the L-19 IVS RNA was confirmedby reacting the enzyme with C₅ p^(*) to form E-p^(*), purifying theE-p^(*), and incubating it with unlabeled C₅ --OH. A labeled productcorresponding to C₅ p^(*) was produced (data not shown). This approachallowed to rapid screening of a series of nucleotides andoligonucleotides for their ability to reverse the phosphorylationreaction. As shown in Table 2, of the oligonucleotides tested only UCUand C₄ U had activity comparable to the of C₅. It remains possible thatsome of the other oligonucleotides have a high K_(m) and would havedetectable activity at a higher concentration

EXAMPLE XII

The L-19 IVS RNA Is a Phosphotransferase. The phosphotransfer reactionsdescribed thus far were all done in enzyme excess. To prove that theL-19 IVS RNA could act catalytically, it was necessary to show that eachenzyme molecule could mediate the dephosphorylation of multiplesubstrate molecules. This was accomplished by the incubation of L-19 IVSRNA with a molar excess of C₅ p^(*) and an even greater molar excess ofunlabeled UCU, to act as a phosphate acceptor. As shown in FIG. 14A,L-19 IVS RNA was capable of transferring the 3'-terminal phosphate fromC₅ p to UCU. Treatment of the product with RNase T2 and thin-layerchromatography confirmed that the phosphate had been transferred from aC to a U residue. The time course in FIG. 14B was done under conditionsof lower enzyme concentration (0.16 uM) and higher acceptorconcentration (200 uM) than those used in the experiment of FIG. 14A.Quantitation of the data (FIG. 14C) showed that under these conditions11 substrate molecules were dephosphorylated per enzyme molecule after120 min. Phosphorylation of the enzyme precedes substantial productformation, consistent with E-p being an oligatory intermediate in thereaction. At steady state 63% of the enzyme is present as E-p.

EXAMPLE XIII Plasmid Construction

Been, Michael D. and Cech, Thomas, R. (1986) Cell 47:207-216 reports theconstruction of the pBG plasmid series. In general, the plasmid used forthese studies (pBGST7) was derived from the cloning vector pUC18. Themethodology for the following manipulations is described in Maniatis,T., et al. (1982) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.: Cold Spring Harbor). pUC18 was partially digested withHaeII and unit length linear molecules were isolated by agarose gelelectrophoresis and electroelution. The recovered DNA was treated withT4 DNA polymerase to make blunt ends and then dephosphorylated with calfintestinal phosphatase. pT7-2 (U.S. Biochemical Corp.) was cut withPvull and HindIII and treated with T4 DNA polymerase, and the fragmentcontaining the phage T7 promoter was isolated by polyacrylamide gelelectrophoresis and electroelution. The promoter-containing fragment wasligated into the linearized pUC18 and the plasmid transformed into E.coli strain JM83. Individual colonies were picked and miniprep DNA wasscreened by restriction endonuclease digestion to locate the promoterfragment to the correct HaeII site (position 680 on the map in the NewEngland Biolabs Catalog). The orientation of the T7 promoter wasdetermined by transcribing EcoRI-cut miniprep DNA with T7 RNA polymeraseand determining the size of the product. This plasmid (pUT718) was thencut in the multicloning site with KpnI and SphI and treated with T4 DNApolymerase followed by calf intestinal phosphatase. An IVS-containingBamHI DNA fragment was isolated from pJE457 (Price, J. V. and Cech, T.R., (1985) Science 228:719-722) and treated with S1 nuclease, phenolextracted, chloroform extracted, and ethanol precipitated. TheS1-treated fragment, containing short deletions at both ends, wasligated into the KpnI/SphI-cut pUT718. E. coli strain JM83 wastransformed with the recombinant plasmid and plated on LB agarcontaining ampicillin and X-gal(5-bromo-4-chloro-3-indolyl-beta-D-galactoside). DNA isolated from bluecolonies was assayed by agarose gels electrophoresis for IVS-sizeinserts. Exon sequences were determined by dideoxy-sequencing theminiprep DNA. Ideally, only plasmids containing IVS and exons that willpreserve the reading frame of the lacZ gene fragment should give bluecolonies and this will depend on the splicing of the message in E. coli(Waring, R. B., et al, (1985) Cell 40:371-380; Price, J. V. and Cech, T.R., (1985) Science 228:719-722). In fact, several plasmids thatconferred a light blue color to the colonies had exons for which correctsplicing of the IVS should not generate the proper reading frame; thesewere not investigated further. One that produced a dark blue colony,pBGST7 (FIG. 16), also had the expected reading frame and was used forthese studies.

Mutagenesis

Oligonucleotide-directed mutagenesis on plasmid DNA was done essentiallyas described by Inouye, S. and Inouye M. (1986) In DNA and RNASynthesis, S. Narang, ed. (New York: Academic Press), in press. pBGST7DNA was cleaved in the gene coding for ampicillin resistance with XmnI.In a separate reaction, pBGST7 DNA was cut with EcoRI and HindIII, sitesthat flank the IVS and exon sequences. The vector portion of the plasmidwas separated from the IVS-containing fragment by agarose gelelectrophoresis and recovered by electroelution. XmnI-cut DNA was mixedwith the vector fragment in the presence of the oligonucleotidecontaining the mismatched base, heated to 95° C. for 5 min, placed at37° C. for 30 min, then at 4° C. for 30 min and then put on ice. Theproducts were treated with dNTPs and Klenow fragment of DNA pol I atroom temperature for 2 hr. E. coli strain JM83 was transformed with theDNA and ampicillin-resistant colonies were picked on the basis of color(white or light blue indicating loss of splicing activity) and theminiprep DNA was sequenced.

The double mutants were made using the plasmid DNA of single mutants andscreening for restored beta-galactosidase activity. pBG/-2G:23C was madefrom pBG/-2G and a second oligonucleotide directed at position 23.pBG/-3G:24C was made starting with pBG/24C and using the oligonucleotidedirected at position -3. pBG/-4G:25C was made by generating a circularheteroduplex from pBG/-4G:25C that had been linearized at differentpositions. Transformation of these heteroduplexes generated blue andlight blue colonies, both at a low frequency. The blue colonies werewild-type sequence the light blues double mutant.

pSPTT1A3:

The ThaI fragment of Tetrahymena ribosomal RNA gene contains the IVS andsmall portions of the flanking exons. Hind III linkers were ligated ontothe ends of the ThaI fragment and it was inserted into the Hind III sitein the polylinker of pSP62 (New England Nuclear), which contains the SP6promoter. The recombinant plasmid was cloned in E. coli by standardmethods.

pT7TT1A3:

Construction of pT7TT1A3 was identical to pSPTT1A3 except that the ThaIfragment was cloned into the Hind III site of pT7-2 (U.S. BiochemicalCorp.) which contains the T7 promoter.

pT7 L-21 plasmid:

pBGST7 (described in Been & Cech, (1986) Cell 47:207 was cleaved withrestriction endonucleases SphI and Hind III and the small fragmentcontaining the IVS minus its first 44 nucleotides was purified. pUC18was cleaved with SpH I and Hind III, mixed with the Sph-HIND IIIfragment from pBGST7 and treated with DNA ligase. E. coli cells weretransformed, colonies were picked, plasmid DNA was isolated fromindividual colonies and sequenced. A plasmid containing the SphI-HindIII fragment of the IVS properly inserted into pUC18 was selected forthe next step. This plasmid was cleaved with SphI and EcoRI restrictionendonucleases and a synthetic oligonucleotide was inserted into theplasmid using DNA ligase. The synthetic oligonucleotide containedone-half of the EcoRI restriction site, the T7 promoter, and nucleotides22-44 of the Tetrahymena IVS, ending at the SphI site. E coli cells weretransformed, colonies were picked, plasmid DNA was isolated fromindividual colonies and sequenced.

The final plasmid pT7L-21 was found by sequence analysis to contain theT7 promoter properly juxtaposed to nucleotide 22 of the IVS. (See FIG.16)

Therefore the method can be used to create defined pieces of RNA. Forexample, these defined pieces can be 5'--OH or 3'--OH end pieces, orcenter pieces containing the active site for study and production ofvarient RNA forms.

                  TABLE 1                                                         ______________________________________                                        Sites of cleavage of large RNA substrates by the                              wild-type L - 19 IVS.sub.β  RNA.                                         Substrate    Site   Size (nt)*                                                                             Sequence+                                        ______________________________________                                        pAK-105 (β-globin pre-mRNA)                                                           1      148                                                                                     ##STR6##                                                     2      464       AACUCU AAGAG                                    pT7-1 (pBR322)                                                                             1      145       UCCCUU UUUUG                                                 2      556       CACUCU CAGUA                                                 3      603       UAUUUU  CUCCU                                                4      805       UCCUCU AGAGU                                    Consensus observed                                                                                          ##STR7##                                        expected                                                                                                    ##STR8##                                        ______________________________________                                         *Size of the GTPlabeled fragment.                                             +Sequences are listed in 5'→3' direction. Arrows indicate sites of     cleavage and guanosine addition.                                               The expected consensus sequence is based on the known sequence at the en     of the 5' exon (CUCUCU) modified by the possibility that either a C or a      at position -5 might be able to pair with the G in the active site. At        position -1, there is some basis for thinking that a C might not be as        good as a U (Davies, R.W., et al. (1982) Nature 300:710-724; Michel, F.,      et al. (1983) EMBO J. 2:33-38; Inoue, T., et al. (1986) J. Mol. Biol.         189:143-165.                                                             

                  TABLE 2                                                         ______________________________________                                        Relative Activity of Different Acceptors in the                               Transphosphorylation Reaction.sup.a                                                  acceptor                                                                             activity                                                        ______________________________________                                               UTP    -                                                                      CTP    -                                                                      UC     -                                                                      CC     -                                                                      AA     -                                                                      GU     -                                                                      UU     -                                                                      CU     -                                                                      AUU    -                                                                      UUU    -                                                                      UCU    ++                                                                     CCU    +/-                                                                    AU.sub.3                                                                             -                                                                      GU.sub.3                                                                             -                                                                      C.sub.4 U                                                                            ++                                                                     C.sub.5                                                                              ++                                                                     U.sub.6                                                                              +/-                                                                    dC.sub.5                                                                             -                                                               ______________________________________                                         .sup.a E-p* was incubated with 10 μM oligonucleotide (or mono or           dinucleotide) in 20 mM MgCl.sub.2 and 50 mM TrisHCl, pH 7.5, at 42.degree     C. for 30 min. Transfer of the phosphate to the oligonucleotide was           assayed by sequencing gel electrophoresis and autoradiography. ++,            approximately the same activity as C.sub.5 ; +/-, barely detectable           activity, estimated as ˜1% that of C.sub.5 ; -, no activity             detectable under these conditions.                                       

What is claimed:
 1. Method for dephosphorylation of an RNA substratecomprising the steps of:providing an enzymatic RNA molecule having adephosphorylation activity independent of any protein, and contactingsaid enzymatic RNA molecule with said RNA substrate under conditionssuitable for said enzymatic RNA molecule to remove the 3' terminalphosphate of said RNA substrate.
 2. A method for locating a nucleotidesequence in an RNA molecule having a first length, comprising the stepsof:providing an enzymatic RNA molecule having an endonuclease activityindependent of any protein, contacting said enzymatic RNA molecule withsaid RNA molecule wherein said enzymatic RNA molecule will cleave saidRNA molecule at any said nucleotide sequence present in said RNAmolecule, and determining the length of resulting RNA moleculesresulting from said contacting step as an indication of the location ofthe nucleotide sequence in said RNA molecule.
 3. A method for locating aplurality of nucleotide sequences in an RNA molecule having a firstlength, comprising the steps of:providing a plurality of differentenzymatic RNA molecules having an endonuclease activity independent ofany protein, each said different enzymatic RNA molecule cleaving saidRNA molecule at a different said nucleotide sequence of said pluralityof nucleotide sequences; contacting said plurality of differentenzymatic RNA molecules with said RNA molecule wherein each saiddifferent enzymatic RNA molecule will cleave said RNA molecule at anysaid different nucleotide sequence present in said RNA molecule, anddetermining the length of resulting RNA molecules resulting from saidcontacting step as an indication of the location of the nucleotidesequence in said RNA molecule.
 4. A method for detecting the presence ofa nucleotide sequence in an RNA molecule, comprising the stepsof:providing an enzymatic RNA molecule having an endonuclease activityindependent of any protein, contacting said enzymatic RNA molecule withsaid RNA molecule wherein said enzymatic RNA molecule will cleave saidRNA molecule of any said nucleotide sequence present in said RNAmolecule to produce two or more fragments of said RNA molecule, anddetecting the presence of said fragments as an indication of thepresence of said nucleotide sequence.
 5. Method of claim 2, 3 or 4wherein a radiolabelled guanosine derivative is provided in saidcontacting step to cause radiolabelling of the 5' end of any cleavedsaid RNA molecule.
 6. Method for producing an enzymatic RNA moleculehaving an endonuclease activity independent of any protein, comprisingthe steps of:(a) transcribing recombinant DNA encoding a precursor RNAmolecule to said enzymatic RNA molecule with an RNA polymerase in vitroto produce said precursor RNA; (b) incubating said precursor RNA underconditions suitable for promoting self-splicing and cyclization of saidprecursor RNA to produce cyclized RNA; (c) contacting said cyclized RNAwith MgCl₂ at pH 9.0 to promote conversion of said cyclized RNA to saidenzymatic RNA molecule; and (d) purifying said enzymatic RNA molecule bypolyacrylamide gel electrophoresis and chromatography.
 7. Method ofclaim 6 wherein said recombinant DNA is cloned in E. coli.
 8. Method ofclaim 6 wherein said recombinant DNA comprises an intron of aTetrahymena gene encoding ribosomal RNA.
 9. Method of claim 6 whereinthe expression of said DNA is regulated by the SP6 or T7 RNA polymerasepromoter.
 10. A method for producing an enzymatic RNA molecule having anendonuclease activity of any protein, comprising the steps of:providingDNA encoding RNA consisting essentially of said enzymatic RNA moleculeunder the transcriptional control of a promoter, and transcribing saidDNA from said promoter to produce said enzymatic RNA molecule.
 11. Avector for production of an enzymatic RNA molecule having anendonuclease activity independent of any protein, comprising:DNAencoding said enzymatic RNA molecule, and a promoter positioned relativeto said DNA to cause transcription of said DNA to form an RNA moleculeconsisting essentially of said enzymatic RNA molecule.
 12. The vector ofclaim 11 wherein said endonuclease activity is specific for a nucleotidesequence defining a cleavage site comprising single stranded RNA in aseparate RNA molecule and causing cleavage at said cleavage site by atransesterification reaction.
 13. The vector of claim 11 wherein saidendonuclease activity is specific for a nucleotide sequence defining acleavage site consisting essentially of a single stranded region of aseparate RNA molecule.
 14. The vector of claim 11 or 13 wherein thenucleotide sequence defining said cleavage site comprises four or morebases.
 15. The vector of claim 13 wherein said endonuclease activitycauses cleavage at said cleavage site by a transesterification reaction.16. The vector of claim 11 wherein said enzymatic RNA molecule comprisesan active site region responsible for said cleavage substantiallyidentical to the active site region of the ribosomal RNA interveningsequence naturally occurring in Tetrahymena.
 17. The vector of claim 16wherein said nucleotide sequence defining said cleavage site is chosenfrom GUCU, CUGU, and CGCU.
 18. The vector of claim 11 wherein saidenzymatic RNA molecule has a pH optimum for said endonuclease activitybetween 7.5 and
 8. 19. The vector of claim 11 wherein said endonucleaseactivity requires the presence of magnesium ions, a denaturant, or aguanine derivative.
 20. The vector of claim 11 wherein said endonucleaseactivity is enhanced by the presence of a divalent cation.
 21. Thevector of claim 20 wherein said divalent cation is magnesium.
 22. Thevector of claim 11 wherein said enzymatic RNA molecule further comprisesa second endonuclease activity, independent of any protein, for nucleicacid comprising deoxyribonucleotides wherein said endonuclease activityand said second endonuclease activity may be the same or different. 23.The vector of claim 11 wherein said enzymatic RNA molecule further has anucleotidyl transferase activity.
 24. The vector of claim 23 whereinsaid nucleotidyl transferase activity causes polymerization ofoligocytidylic acid to polycytidylic acid.
 25. The vector of claim 11wherein said enzymatic RNA molecule further has a dephosphorylaseactivity.
 26. The vector of claim 25 wherein said dephosphorylaseactivity causes removal of a 3' terminal phosphate of a nucleic acidcomprising a 3' ribonucleotide.
 27. The vector of claim 26 wherein saiddephosphorylase activity is specific to a specific nucleotide sequence.28. The vector of claim 11 wherein said enzymatic RNA molecule comprisesthe ribonucleotide base sequence of L-19 RNA, said enzymatic RNAmolecule having a ribonucleotide base sequence at the active siteresponsible for said cleavage different from that of L-19 fromTetrahymena.
 29. The vector of claim 11 wherein said enzymatic RNAmolecule comprises the ribonucleotide base sequence of L-19 RNA.
 30. Thevector of claim 29 wherein said L-19 comprises bases 19-331 ofTetrahymena L-IVS.
 31. The vector of claim 11 wherein said activitycauses cleavage of a separate RNA molecule at a specific nucleotidesequence defining a cleavage site comprising single stranded RNA in aseparate RNA molecule, said enzymatic RNA molecule not causing specificcleavage of said separate RNA molecule immediately adjacent in thedirection 3' to the nucleotide sequence CUCU.
 32. The vector of claim11, said vector being a plasmid.
 33. The vector of claim 11 wherein saidpromoter is selected from the group consisting of the bacteriophage SP6and bacteriophage T7 promoters.
 34. The vector of claim 11, wherein saidenzymatic RNA molecule comprises bases 21-331 of Tetrahymena L-IVS. 35.A plasmid selected from the group consisting of pSPTT1A3, pT7-TT1A3,pBGST7, pBG/-2G:23C, pBG/23C, pBG/-3G:24C, pBG/24C, pBG/-4G:25C,pBG/25C, and pBG/23A₄.